Search

Context: Central congenital hypothyroidism (CCH) is an underdiagnosed disorder characterized by deficient production and bioactivity of thyroid-stimulating hormone (TSH) leading to low thyroid hormone synthesis. Thyrotropin-releasing hormone (TRH) receptor (TRHR) defects are rare recessive disorders usually associated with incidentally identified CCH and short stature in childhood., Objectives: Clinical and genetic characterization of a consanguineous family of Roma origin with central hypothyroidism and identification of underlying molecular mechanisms., Design: All family members were phenotyped with thyroid hormone profiles, pituitary magnetic resonance imaging, TRH tests, and dynamic tests for other pituitary hormones. Candidate TRH, TRHR, TSHB, and IGSF1 genes were screened for mutations. A mutant TRHR was characterized in vitro and by molecular modeling., Results: A homozygous missense mutation in TRHR (c.392T > C; p.I131T) was identified in an 8-year-old boy with moderate hypothyroidism (TSH: 2.61 mIU/L, Normal: 0.27 to 4.2; free thyroxine: 9.52 pmol/L, Normal: 10.9 to 25.7) who was overweight (body mass index: 20.4 kg/m2, p91) but had normal stature (122 cm; -0.58 standard deviation). His mother, two brothers, and grandmother were heterozygous for the mutation with isolated hyperthyrotropinemia (TSH: 4.3 to 8 mIU/L). The I131T mutation, in TRHR intracellular loop 2, decreases TRH affinity and increases the half-maximal effective concentration for signaling. Modeling of TRHR-Gq complexes predicts that the mutation disrupts the interaction between receptor and a hydrophobic pocket formed by Gq., Conclusions: A unique missense TRHR defect identified in a consanguineous family is associated with central hypothyroidism in homozygotes and hyperthyrotropinemia in heterozygotes, suggesting compensatory elevation of TSH with reduced biopotency. The I131T mutation decreases TRH binding and TRHR-Gq coupling and signaling., (Copyright © 2017 Endocrine Society)

Transport of bile acids across the basolateral membrane of the intestinal enterocyte is carried out by the organic solute transporter (Ost) composed of a seven-transmembrane domain (TMD) subunit (Ostα) and an ancillary single TMD subunit (Ostβ). Although previous investigations have demonstrated the importance of the TMD of Ostβ for its activity, further studies were conducted to assess the contributions of other regions of the Ostβ subunit. Transport activity was retained when Ostβ was truncated to contain only the TMD with 15 additional residues on each side and co-expressed with Ostα, whereas shorter fragments were inactive. To probe the broader functions of Ostβ segments, chimeric proteins were constructed in which N-terminal, TMD or C-terminal regions of Ostβ were fused to corresponding regions of receptor activity-modifying protein (RAMP1), a single TMD protein required by several seven-TMD G-protein-coupled receptors including the calcitonin receptor-like receptor (CLR). Ostβ/RAMP1 chimeras were expressed with Ostα and CLR. As expected, replacing the Ostβ TMD abolished transport activity; however, replacing either the entire N-terminal or entire C-terminal domain of Ostβ with RAMP1 sequences did not prevent plasma membrane localization or the ability to support [ 3 H]taurocholate uptake. Co-immunoprecipitation experiments revealed that the C-terminus of Ostβ is a previously unrecognized site of interaction with Ostα. All chimeras containing N-terminal RAMP1 segments allowed co-expressed CLR to respond to agonists with strong increases in cyclic AMP. These results provide new insights into the structure and function of the heteromeric Ost transporter complex., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

IGSF1 (Immunoglobulin Superfamily 1) gene defects cause central hypothyroidism and macroorchidism. However, the pathogenic mechanisms of the disease remain unclear. Based on a patient with a full deletion of IGSF1 clinically followed from neonate to adulthood, we investigated a common pituitary origin for hypothyroidism and macroorchidism, and the role of IGSF1 as regulator of pituitary hormone secretion. The patient showed congenital central hypothyroidism with reduced TSH biopotency, over-secretion of FSH at neonatal minipuberty and macroorchidism from 3 years of age. His markedly elevated inhibin B was unable to inhibit FSH secretion, indicating a status of pituitary inhibin B resistance. We show here that IGSF1 is expressed both in thyrotropes and gonadotropes of the pituitary and in Leydig and germ cells in the testes, but at very low levels in Sertoli cells. Furthermore, IGSF1 stimulates transcription of the thyrotropin-releasing hormone receptor (TRHR) by negative modulation of the TGFβ1-Smad signaling pathway, and enhances the synthesis and biopotency of TSH, the hormone secreted by thyrotropes. By contrast, IGSF1 strongly down-regulates the activin-Smad pathway, leading to reduced expression of FSHB, the hormone secreted by gonadotropes. In conclusion, two relevant molecular mechanisms linked to central hypothyroidism and macroorchidism in IGSF1 deficiency are identified, revealing IGSF1 as an important regulator of TGFβ/Activin pathways in the pituitary.

Melanocortin 2 receptor accessory protein (MRAP) facilitates trafficking of melanocortin 2 (MC2) receptors and is essential for ACTH binding and signaling. MRAP is a single transmembrane domain protein that forms antiparallel homodimers. These studies ask when MRAP first acquires this dual topology, whether MRAP architecture is static or stable, and whether the accessory protein undergoes rapid turnover. To answer these questions, we developed an approach that capitalizes on the specificity of bacterial biotin ligase, which adds biotin to lysine in a short acceptor peptide sequence; the distinct mobility of MRAP protomers of opposite orientations based on their N-linked glycosylation; and the ease of identifying biotin-labeled proteins. We inserted biotin ligase acceptor peptides at the N- or C-terminal ends of MRAP and expressed the modified proteins in mammalian cells together with either cytoplasmic or endoplasmic reticulum-targeted biotin ligase. MRAP assumed dual topology early in biosynthesis in both CHO and OS3 adrenal cells. Once established, MRAP orientation was stable. Despite its conformational stability, MRAP displayed a half-life of under 2 h in CHO cells. The amount of MRAP was increased by the proteasome inhibitor MG132 and MRAP underwent ubiquitylation on lysine and other amino acids. Nonetheless, when protein synthesis was blocked with cycloheximide, MRAP was rapidly degraded even when MG132 was included and all lysines were replaced by arginines, implicating non-proteasomal degradation pathways. The results show that although MRAP does not change orientations during trafficking, its synthesis and degradation are dynamically regulated.

In many vertebrate species visible melanin-based pigmentation patterns correlate with high stress- and disease-resistance, but proximate mechanisms for this trait association remain enigmatic. Here we show that a missense mutation in a classical pigmentation gene, melanocyte stimulating hormone receptor (MC1R), is strongly associated with distinct differences in steroidogenic melanocortin 2 receptor (MC2R) mRNA expression between high- (HR) and low-responsive (LR) rainbow trout (Oncorhynchus mykiss). We also show experimentally that cortisol implants increase the expression of agouti signaling protein (ASIP) mRNA in skin, likely explaining the association between HR-traits and reduced skin melanin patterning. Molecular dynamics simulations predict that melanocortin 2 receptor accessory protein (MRAP), needed for MC2R function, binds differently to the two MC1R variants. Considering that mRNA for MC2R and the MC1R variants are present in head kidney cells, we hypothesized that MC2R activity is modulated in part by different binding affinities of the MC1R variants for MRAP. Experiments in mammalian cells confirmed that trout MRAP interacts with the two trout MC1R variants and MC2R, but failed to detect regulation of MC2R signaling, possibly due to high constitutive MC1R activity.

The melanocortin-2 (MC2) receptor is a G protein-coupled receptor that mediates responses to ACTH. The MC2 receptor acts in concert with the MC2 receptor accessory protein (MRAP) that is absolutely required for ACTH binding and signaling. MRAP has a single transmembrane domain and forms a highly unusual antiparallel homodimer that is stably associated with MC2 receptors at the plasma membrane. Despite the physiological importance of the interaction between the MC2 receptor and MRAP, there is little understanding of how the accessory protein works. The dual topology of MRAP has made it impossible to determine whether highly conserved and necessary regions of MRAP are required on the intracellular or extracellular face of the plasma membrane. The strategy used here was to fix the orientation of two antiparallel MRAP molecules and then introduce inactivating mutations on one side of the membrane or the other. This was achieved by engineering proteins containing tandem copies of MRAP fused to the amino terminus of the MC2 receptor. The data firmly establish that only the extracellular amino terminus (Nout) copy of MRAP, oriented with critical segments on the extracellular side of the membrane, is essential. The transmembrane domain of MRAP is also required in only the Nout orientation. Finally, activity of MRAP-MRAP-MC2-receptor fusion proteins with inactivating mutations in either MRAP or the receptor was rescued by co-expression of free wild-type MRAP or free wild-type receptor. These results show that the basic MRAP-MRAP-receptor signaling unit forms higher order complexes and that these multimers signal., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Organic solute transporterα-OSTβ is a bile acid transporter important for bile acid recycling in the enterohepatic circulation. In comparison to wild-type mice, Ostα(-/-) mice have a lower bile acid pool and increased fecal lipids and they are relatively resistant to age-related weight gain and insulin resistance. These studies tested whether Ostα(-/-) mice are also protected from weight gain, lipid changes, and insulin resistance which are normally observed with a western-style diet high in both fat and cholesterol (WD). Wild-type and Ostα(-/-) mice were fed a WD, a control defined low-fat diet (LF) or standard laboratory chow (CH). Surprisingly, although the Ostα(-/-) mice remained lighter on LF and CH diets, they weighed the same as wild-type mice after 12 weeks on the WD even though bile acid pool levels remained low and fecal lipid excretion remained elevated. Mice of both genotypes excreted relatively less lipid when switched from CH to LF or WD. WD caused slightly greater changes in expression of genes involved in lipid transport in the small intestines of Ostα(-/-) mice than wild-type, but the largest differences were between CH and defined diets. After WD feeding, Ostα(-/-) mice had lower serum cholesterol and hepatic lipids, but Ostα(-/-) and wild-type mice had equivalent levels of muscle lipids and similar responses in glucose and insulin tolerance tests. Taken together, the results show that Ostα(-/-) mice are able to adapt to a western-style diet despite low bile acid levels., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

The organic solute transporter OSTα-OSTβ is a key transporter for the efflux of bile acids across the basolateral membrane of ileocytes and the subsequent return of bile acids to the liver. Ostα(-/-) mice exhibit reduced bile acid pools and impaired lipid absorption. In this study, wild-type and Ostα(-/-) mice were characterized at 5 and 12 mo of age. Ostα(-/-) mice were resistant to age-related weight gain, body fat accumulation, and liver and muscle lipid accumulation, and male Ostα(-/-) mice lived slightly longer than wild-type mice. Caloric intake and activity levels were similar for Ostα(-/-) and wild-type male mice. Fecal lipid excretion was increased in Ostα(-/-) mice, indicating that a defect in lipid absorption contributes to decreased fat accumulation. Analysis of genes involved in intestinal lipid absorption revealed changes consistent with decreased dietary lipid absorption in Ostα(-/-) animals. Hepatic expression of cholesterol synthetic genes was upregulated in Ostα(-/-) mice, showing that increased cholesterol synthesis partially compensated for reduced dietary cholesterol absorption. Glucose tolerance was improved in male Ostα(-/-) mice, and insulin sensitivity was improved in male and female Ostα(-/-) mice. Akt phosphorylation was measured in liver and muscle tissue from mice after acute administration of insulin. Insulin responses were significantly larger in male and female Ostα(-/-) than wild-type mice. These findings indicate that loss of OSTα-OSTβ protects against age-related weight gain and insulin resistance.

The melanocortin-4 receptor (MC4R) is essential for control of energy homeostasis in vertebrates. MC4R interacts with melanocortin receptor accessory protein 2 (MRAP2) in vitro, but its functions in vivo are unknown. We found that MRAP2a, a larval form, stimulates growth of zebrafish by specifically blocking the action of MC4R. In cell culture, this protein binds MC4R and reduces the ability of the receptor to bind its ligand, α-melanocyte-stimulating hormone (α-MSH). A paralog, MRAP2b, expressed later in development, also binds MC4R but increases ligand sensitivity. Thus, MRAP2 proteins allow for developmental control of MC4R activity, with MRAP2a blocking its function and stimulating growth during larval development, whereas MRAP2b enhances responsiveness to α-MSH once the zebrafish begins feeding, thus increasing the capacity for regulated feeding and growth.

G protein-coupled receptors (GPCRs) are an ubiquitously expressed class of transmembrane proteins involved in the signal transduction of neurotransmitters, hormones and various other ligands. Their signaling output is desensitized by mechanisms involving phosphorylation, internalization, and dissociation from G proteins and resensitized by mechanisms involving dephosphorylation, but details about the phosphatases responsible are generally lacking. We describe here the use of an siRNA-based library to knock down expression of specific phosphatase subunits to identify protein phosphatase 1-α (PP1α) as important for the thyrotropin-releasing hormone (TRH) receptor. Inhibition of PP1α synthesis and overexpression of dominant negative PP1α preserved receptor phosphorylation under conditions favoring dephosphorylation, whereas overexpression of PP1α accelerated dephosphorylation. Knockdown of all three PP1 catalytic subunits inhibited TRH receptor phosphorylation much more powerfully than knockdown of PP1α alone, suggesting that different PP1 isoforms function redundantly. Knockdown of a structural subunit of PP2A, a second potential hit in the library screen, was ineffective. Calyculin A, a potent inhibitor of PP1 family phosphatases, strongly inhibited dephosphorylation of transfected TRH receptors and endogenous receptors in pituitary cells, but fostriecin, which is selective for PP2A family phosphatases, did not. We conclude that the PP1 class of phosphatases is essential for TRH receptor dephosphorylation.

The pituitary receptor for thyrotropin-releasing hormone (TRH) is a calcium-mobilizing G protein-coupled receptor (GPCR) that signals through Gq/11, elevating calcium, and activating protein kinase C. TRH receptor signaling is quickly desensitized as a consequence of receptor phosphorylation, arrestin binding, and internalization. Following activation, TRH receptors are phosphorylated at multiple Ser/Thr residues in the cytoplasmic tail. Phosphorylation catalyzed by GPCR kinase 2 (GRK2) takes place rapidly, reaching a maximum within seconds. Arrestins bind to two phosphorylated regions, but only arrestin bound to the proximal region causes desensitization and internalization. Phosphorylation at Thr365 is critical for these responses. TRH receptors internalize in clathrin-coated vesicles with bound arrestin. Following endocytosis, vesicles containing phosphorylated TRH receptors soon merge with rab5-positive vesicles. Over approximately 20 min these form larger endosomes rich in rab4 and rab5, early sorting endosomes. After TRH is removed from the medium, dephosphorylated receptors start to accumulate in rab4-positive, rab5-negative recycling endosomes. The mechanisms responsible for sorting dephosphorylated receptors to recycling endosomes are unknown. TRH receptors from internal pools help repopulate the plasma membrane. Dephosphorylation of TRH receptors begins when TRH is removed from the medium regardless of receptor localization, although dephosphorylation is fastest when the receptor is on the plasma membrane. Protein phosphatase 1 is involved in dephosphorylation but the details of how the enzyme is targeted to the receptor remain obscure. It is likely that future studies will identify biased ligands for the TRH receptor, novel arrestin-dependent signaling pathways, mechanisms responsible for targeting kinases and phosphatases to the receptor, and principles governing receptor trafficking.

G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. Herein we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine-Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.

The organic solute transporter, Ost/Slc51, is composed of two distinct proteins that must heterodimerize to generate transport activity, but the role of the individual subunits in mediating transport activity is unknown. The present study identified regions in Ostβ required for heterodimerization with Ostα, trafficking of the Ostα-Ostβ complex to the plasma membrane, and bile acid transport activity in HEK293 cells. Bimolecular fluorescence complementation analysis revealed that a 25-amino acid peptide containing the Ostβ transmembrane (TM) domain heterodimerized with Ostα, although the resulting complex failed to reach the plasma membrane and generate cellular [(3)H]taurocholate transport activity. Deletion of the single TM domain of Ostβ abolished interaction with Ostα, demonstrating that the TM segment is necessary and sufficient for formation of a heteromeric complex with Ostα. Mutation of the highly conserved tryptophan-asparagine sequence within the TM domain of Ostβ to alanines did not prevent cell surface trafficking, but abolished transport activity. Removal of the N-terminal 27 amino acids of Ostβ resulted in a transporter complex that reached the plasma membrane and exhibited transport activity at 30 °C. Complete deletion of the C terminus of Ostβ abolished [(3)H]taurocholate transport activity, but reinsertion of two native arginines immediately C-terminal to the TM domain rescued this defect. These positively charged residues establish the correct N(exo)/C(cyt) topology of the peptide, in accordance with the positive inside rule. Together, the results demonstrate that Ostβ is required for both proper trafficking of Ostα and formation of the functional transport unit, and identify specific residues of Ostβ critical for these processes.

Analysis of the functional expression of the melanocortin 2 receptor (MC2R) from a rather broad spectrum of vertebrates indicates that MC2R is exclusively selective for the ligand, ACTH, and the melanocortin receptor accessory protein 1 (MRAP1) is required for high affinity ACTH binding and activation of MC2R. A phylogenetic analysis of MRAP1 suggested that tetrapod sequences and bony fish sequences may represent two distinct trends in the evolution of the mrap1 gene. To test this hypothesis, a frog (Xenopus tropicalis) MC2R was expressed in CHO cells either in the presence of a tetrapod (mouse) MRAP1 or a bony fish (zebrafish) MRAP1. The response of frog MC2R to different concentrations of human ACTH(1-24) was more robust in the presence of mouse MRAP1 than in the presence of zebrafish MRAP1. Conversely, the cAMP response mediated by the rainbow trout (Oncorhynchus mykiss) MC2R was almost twofold higher and occurred at 1000-fold lower ACTH concentration in the presence of zebrafish MRAP1 than in the presence of mouse MRAP1. Collectively, these experiments raise the possibility that at least two distinct trends have emerged in the co-evolution of MC2R/MRAP1 interactions during the radiation of the vertebrates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The melanocortin 2 (MC(2)) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC(2) receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC(2) receptor responsible for these characteristics, we created chimeras between MC(2) and MC(4) receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC(2) but not MC(4) receptors. Replacing the first transmembrane segment of the MC(2) receptor with the corresponding region from the MC(4) receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC(4) receptor with the corresponding regions from the MC(2) receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC(2) receptors with MC(4) sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-d-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC(2) receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second to third transmembrane segments of the MC(2) receptor as important for MRAP dependent-signal transduction and ligand specificity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Two GPCRs (G-protein-coupled receptors), TRHR (thyrotropin-releasing hormone receptor) and beta(2)AR (beta(2)-adrenergic receptor), are regulated in distinct manners. Following agonist binding, TRHR undergoes rapid phosphorylation attributable to GRKs (GPCR kinases); beta(2)AR is phosphorylated by both second messenger-activated PKA (protein kinase A) and GRKs with slower kinetics. TRHR co-internalizes with arrestin, whereas beta(2)AR recruits arrestin, but internalizes without it. Both receptors are dephosphorylated following agonist removal, but TRHR is dephosphorylated much more rapidly while it remains at the plasma membrane. We generated chimaeras swapping the C-terminal domains of these receptors to clarify the role of different receptor regions in phosphorylation, internalization and dephosphorylation. beta(2)AR with a TRHR cytoplasmic tail (beta(2)AR-TRHR) and TRHR with a beta(2)AR tail (TRHR-beta(2)AR) signalled to G-proteins normally. beta(2)AR-TRHR was phosphorylated well at the PKA site in the third intracellular loop, but poorly at GRK sites in the tail, whereas TRHR-beta(2)AR was phosphorylated strongly at GRK sites in the tail (Ser(355)/Ser(356) of the beta(2)AR). Both chimaeric receptors exhibited prolonged, but weak, association with arrestin at the plasma membrane, but high-affinity arrestin interactions and extensive co-internalization of receptor with arrestin required a phosphorylated TRHR tail. In contrast, swapping C-terminal domains did not change the rates of phosphorylation and dephosphorylation or the dependence of TRHR dephosphorylation on the length of agonist exposure. Thus the interactions of GPCRs with GRKs and phosphatases are determined not simply by the amino acid sequences of the substrates, but by regions outside the cytoplasmic tails.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs), which constitute the largest family of membrane proteins, mediate responses to diverse physiological stimuli. The presence of melanocortin 2 receptors (MC2Rs) on the plasma membrane requires the presence of either MC2R accessory protein (MRAP) or MRAP2, which are homologous accessory proteins. Here, we show that, whereas MRAP was essential for activation of MC2R signaling, MRAP2 was an endogenous inhibitor that competed with MRAP for binding to MC2R and decreased the potency of adrenocorticotropic hormone (ACTH), the endogenous agonist for MC2Rs, in stimulating the production of adenosine 3',5'-monophosphate (cAMP). ACTH bound with high affinity to MC2Rs in the presence of MRAP, but not MRAP2. The ability of MRAP and MRAP2 to influence ligand-binding affinity was specific to MC2R, because these proteins had little effect on the binding of NDP-alpha-melanocyte-stimulating hormone to MC4R or on its stimulation of cAMP responses. These results demonstrate that the balance of stimulatory and inhibitory accessory proteins can control the sensitivity of a GPCR to its natural agonist.

The thyrotropin-releasing hormone (TRH) receptor undergoes rapid and extensive agonist-dependent phosphorylation attributable to G protein-coupled receptor (GPCR) kinases (GRKs), particularly GRK2. Like many GPCRs, the TRH receptor is predicted to form an amphipathic helix, helix 8, between the NPXXY motif at the cytoplasmic end of the seventh transmembrane domain and palmitoylation sites at Cys335 and Cys337. Mutation of all six lysine and arginine residues between the NPXXY and residue 340 to glutamine (6Q receptor) did not prevent the receptor from stimulating inositol phosphate turnover but almost completely prevented receptor phosphorylation in response to TRH. Phosphorylation at all sites in the cytoplasmic tail was inhibited. The phosphorylation defect was not reversed by long incubation times or high TRH concentrations. As expected for a phosphorylation-defective receptor, the 6Q-TRH receptor did not recruit arrestin, undergo the typical arrestin-dependent increase in agonist affinity, or internalize well. Lys326, directly before phenylalanine in the common GPCR motif NPXXY(X)(5-6)F(R/K), was critical for phosphorylation. The 6Q-TRH receptor was not phosphorylated effectively in cells overexpressing GRK2 or in in vitro kinase assays containing purified GRK2. Phosphorylation of the 6Q receptor was partially restored by coexpression of a receptor with an intact helix 8 but without phosphorylation sites. Phosphorylation was inhibited but not completely prevented by alanine substitution for cysteine palmitoylation sites. Positively charged amino acids in the proximal tail of the beta2-adrenergic receptor were also important for GRK-dependent phosphorylation. The results indicate that positive residues in helix 8 of GPCRs are important for GRK-dependent phosphorylation.

Activation of the G protein-coupled TRH receptor leads to its phosphorylation and internalization. These studies addressed the fundamental question of whether phosphorylation regulates receptor trafficking or endosomal localization regulates the phosphorylation state of the receptor. Trafficking of phosphorylated and dephosphorylated TRH receptors was characterized using phosphosite-specific antibody after labeling surface receptors with antibody to an extracellular epitope tag. Rab5 and phosphoreceptor did not colocalize at the plasma membrane immediately after TRH addition but overlapped extensively by 15 min. Dominant-negative Rab5-S34N inhibited receptor internalization. Later, phosphoreceptor was in endosomes containing Rab5 and Rab4. Dephosphorylated receptor colocalized with Rab4 but not with Rab5. Dominant-negative Rab4, -5, or -11 did not affect receptor phosphorylation or dephosphorylation, showing that phosphorylation determines localization in Rab4(+)/Rab5(-) vesicles and not vice versa. No receptor colocalized with Rab7; a small amount of phosphoreceptor colocalized with Rab11. To characterize recycling, surface receptors were tagged with antibody, or surface receptors containing an N-terminal biotin ligase acceptor sequence were labeled with biotin. Most recycling receptors did not return to the plasma membrane for more than 2 h after TRH was removed, whereas the total cell surface receptor density was largely restored in less than 1 h, indicating that recruited receptors contribute heavily to early repopulation of the plasma membrane.

MC2 (ACTH) receptors require MC2 receptor accessory protein (MRAP) to reach the cell surface. In this study, we show that MRAP has the opposite effect on the closely related MC5 receptor. In enzyme-linked immunosorbent assay and microscopy experiments, MC2 receptor was retained in the endoplasmic reticulum in the absence of MRAP and targeted to the plasma membrane with MRAP. MC5 receptor was at the plasma membrane in the absence of MRAP, but trapped intracellularly when expressed with MRAP. Using bimolecular fluorescence complementation, where one fragment of yellow fluorescent protein (YFP) was fused to receptors and another to MRAP, we showed that MC2 receptor-MRAP dimers were present at the plasma membrane, whereas MC5 receptor-MRAP dimers were intracellular. Both MC2 and MC5 receptors co-precipitated with MRAP. MRAP did not alter expression of beta2-adrenergic receptors or co-precipitate with them. To determine if MRAP affects formation of receptor oligomers, we co-expressed MC2 receptors fused to YFP fragments in the presence or absence of MRAP. YFP fluorescence, reporting MC2 receptor homodimers, was readily detectable with or without MRAP. In contrast, MC5 receptor homodimers were visible in the absence of MRAP, but little fluorescence was observed by microscopic analysis when MRAP was co-expressed. Co-precipitation of differentially tagged receptors confirmed that MRAP blocks MC5 receptor dimerization. The regions of MRAP required for its effects on MC2 and MC5 receptors differed. These results establish that MRAP forms stable complexes with two different melanocortin receptors, facilitating surface expression of MC2 receptor but disrupting dimerization and surface localization of MC5 receptor.

The melanocortin2 (MC2), or ACTH receptor, requires MC2 receptor accessory protein (MRAP) for function, and individuals lacking MRAP are ACTH-resistant and glucocorticoid-deficient. MRAP facilitates trafficking of the MC2 receptor to the plasma membrane and is absolutely required for ACTH binding and stimulation of cAMP. MRAP, which contains a single transmembrane domain, has a unique structure, an antiparallel homodimer. It can be isolated from the plasma membrane in a complex with the MC2 receptor. A short sequence just aminoterminal to the transmembrane domain of MRAP is essential for dual topology, while the transmembrane region is not; both are necessary for function. Deletion or alanine-substitution of other aminoterminal regions yields MRAP mutants that promote surface expression of the MC2 receptor but not receptor signaling. These results identify two distinct actions of MRAP: to permit trafficking of the MC2 receptor, and to allow surface receptor binding and signaling.

MRAP, melanocortin 2 (MC2) receptor accessory protein, is required for trafficking by the MC2 (ACTH) receptor. MRAP is a single transmembrane protein that forms highly unusual antiparallel homodimers. We used molecular complementation to ask where MRAP achieves dual topology. Fragments of yellow fluorescent protein (YFP) were fused to the NH2 or COOH terminus of MRAP such that YFP fluorescence could occur only in antiparallel homodimers; fluorescence was present in the endoplasmic reticulum. MRAP retained dual topology after deletion of most of the amino terminus. In contrast, deletion of residues 31-37, just NH2-terminal to the transmembrane domain, forced MRAP into a single Nexo/Ccyt orientation and blocked its ability to promote MC2 receptor trafficking and homodimerize. When the transmembrane domain of MRAP was replaced with the corresponding region from RAMP3, dual topology was retained but MRAP was inactive. Insertion of MRAP residues 29-37 conferred dual topology to RAMP3, normally in an Nexo/Ccyt orientation. When expressed with MRAPDelta1-30, MRAPDelta10-20, or MRAPDelta21-30, MC2 receptor was localized on the plasma membrane but unable to respond to ACTH. Residues 18-21 of MRAP were critical; MC2 receptor expressed with MRAP(18-21A) localized to the plasma membrane but did not bind 125I-ACTH or increase cAMP in response to ACTH. A newly identified MRAP homolog, MRAP2, lacks amino acids 18LDYI21 of MRAP and, like MRAP(18-21A), allows MC2 receptor trafficking but not signaling. MRAP2 with an LDYI insertion functions like MRAP. These results demonstrate that MRAP not only facilitates MC2 receptor trafficking but also allows properly localized receptor to bind ACTH and consequently signal.

Arrestin binding to agonist-occupied phosphorylated G protein-coupled receptors typically increases the affinity of agonist binding, increases resistance of receptor-bound agonist to removal with high acid/salt buffer, and leads to receptor desensitization and internalization. We tested whether thyrotropin-releasing hormone (TRH) receptors lacking phosphosites in the C-terminal tail could form stable and functional complexes with arrestin. Fibroblasts from mice lacking arrestins 2 and 3 were used to distinguish between arrestin-dependent and -independent effects. Arrestin did not promote internalization or desensitization of a receptor that had key Ser/Thr phosphosites mutated to Ala (4Ala receptor). Nevertheless, arrestin greatly increased acid/salt resistance and the affinity of 4Ala receptor for TRH. Truncation of 4Ala receptor just distal to the key phosphosites (4AlaStop receptor) abolished arrestin-dependent acid/salt resistance but not the effect of arrestin on agonist affinity. Arrestin formed stable complexes with activated wild-type and 4Ala receptors but not with 4AlaStop receptor, as measured by translocation of arrestin-green fluorescent protein to the plasma membrane or chemical cross-linking. An arrestin mutant that does not interact with clathrin and AP2 did not internalize receptor but still promoted high affinity TRH binding, acid/salt resistance, and desensitization. A sterically restricted arrestin mutant did not cause receptor internalization or desensitization but did promote acid/salt resistance and high agonist affinity. The results demonstrate that arrestin binds to proximal or distal phosphosites in the receptor tail. Arrestin binding at either site causes increased agonist affinity and acid/salt resistance, but only the proximal phosphosites evoke the necessary conformational changes in arrestin for receptor desensitization and internalization.

Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all-L counterpart (Glp-Asn-Pro-Tyr-TrpNH(2)) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [(3)H][3-Me-His(2)]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His(2)]TRH potently displaced [(3)H][3-Me-His(2)]TRH binding from all tissues/cells investigated. Glp-Asn-Pro-D-Tyr-D-TrpNH(2) in concentrations up to 10(-5)M did not displace [(3)H][3-Me-His(2)]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all-L peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.7+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2), analogous to cortical membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.8+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2). Neither peptide, however, displaced [(3)H][3-Me-His(2)]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is not displacing [(3)H][3-Me-His(2)]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.

The melanocortin-2 (MC2) receptor accessory protein (MRAP) is required for trafficking of the G protein-coupled MC2 receptor to the plasma membrane. The mechanism of action and structure of MRAP, which has a single transmembrane domain, are unknown. Here, we show that MRAP displays a previously uncharacterized topology. Epitopes on both the N- and C-terminal ends of MRAP were localized on the external face of CHO cells at comparable levels. Using antibodies raised against N- and C-terminal MRAP peptides, we demonstrated that both ends of endogenous MRAP face the outside in adrenal cells. Nearly half of MRAP was glycosylated at the single endogenous N-terminal glycosylation site, and over half was glycosylated when the natural glycosylation site was replaced by one in the C-terminal domain. A mutant MRAP with potential glycosylation sites on both sides of the membrane was singly but not doubly glycosylated, suggesting that MRAP is not monotopic. Coimmunoprecipitation of differentially tagged MRAPs established that MRAP is a dimer. By selectively immunoprecipitating cell surface MRAP in one or the other orientation, we showed that MRAP homodimers are antiparallel and form a stable complex with MC2 receptor. In the absence of MRAP, MC2 receptor was trapped in the endoplasmic reticulum, but with MRAP, the MC2 receptor was glycosylated and localized on the plasma membrane, where it signaled in response to ACTH. MRAP acted specifically, because it did not increase surface expression of other melanocortin, beta2-adrenergic, or TSH-releasing hormone receptors. MRAP is the first eukaryotic membrane protein identified with an antiparallel homodimeric structure.

The G protein-coupled thyrotropin (TSH)-releasing hormone (TRH) receptor forms homodimers. Regulated receptor dimerization increases TRH-induced receptor endocytosis. These studies test whether dimerization increases receptor phosphorylation, which could potentiate internalization. Phosphorylation at residues 355-365, which is critical for internalization, was measured with a highly selective phospho-site-specific antibody. Two strategies were used to drive receptor dimerization. Dimerization of a TRH receptor-FK506-binding protein (FKBP) fusion protein was stimulated by a dimeric FKBP ligand. The chemical dimerizer caused a large increase in TRH-dependent phosphorylation within 1 min, whereas a monomeric FKBP ligand had no effect. The dimerizer did not alter phoshorylation of receptors lacking the FKBP domain. Dimerization of receptors containing an N-terminal HA epitope also was induced with anti-HA antibody. Anti-HA IgG strongly increased TRH-induced phosphorylation, whereas monomeric Fab fragments had no effect. Anti-HA antibody did not alter phosphorylation in receptors lacking an HA tag. Furthermore, two phosphorylation-defective TRH receptors functionally complemented one another and permitted phosphorylation. Receptors with a D71A mutation in the second transmembrane domain do not signal, whereas receptors with four Ala mutations in the 355-365 region signal normally but lack phosphorylation sites. When D71A- and 4Ala-TRH receptors were expressed alone, neither underwent TRH-dependent phosphorylation. When they were expressed together, D71A receptor was phosphorylated by G protein-coupled receptor kinases in response to TRH. These results suggest that the TRH receptor is phosphorylated preferentially when it is in dimers or when preexisting receptor dimers are driven into microaggregates. Increased receptor phosphorylation may amplify desensitization.

Thyrotropin-releasing hormone (TRH) displays multiple CNS-mediated actions that have long been recognized to have therapeutic potential in treating a wide range of neurological disorders. Investigations of CNS functions and clinical use of TRH are hindered, however, due to its rapid degradation by TRH-degrading ectoenzyme (TRH-DE). We now report the discovery of a set of first-in-class compounds that display unique ability to both potently inhibit TRH-DE and bind to central TRH receptors with unparalleled affinity. This dual pharmacological activity within one molecular entity was found through selective manipulation of peptide stereochemistry. Notably, the lead compound of this set, L-pyroglutamyl-L-asparaginyl-L-prolyl-D-tyrosyl-D-tryptophan amide (Glp-Asn-Pro-D-Tyr-D-TrpNH(2)), is effective in vivo at producing and potentiating central actions of TRH without evoking release of thyroid-stimulating hormone (TSH). Specifically, this peptide displayed high plasma stability and combined potent inhibition of TRH-DE (K(i) 151 nM) with high affinity binding to central TRH receptors (K(i) 6.8 nM). Moreover, intraperitoneal injection of this peptide mimicked and augmented the effects of TRH on behavioural activity in rat. Analogous to TRH, it also antagonized pentobarbital-induced narcosis when administered intravenously. This discovery provides new opportunities for probing the role of TRH actions in the CNS and a basis for development of novel TRH-based neurotherapeutics.

To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr(365) in the receptor tail; distal sites were more phosphorylated in some heterologous models. beta-Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nm TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t((1/2)) approximately 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t((1/2)) approximately 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin- and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.

The melanocortin-4 (MC4) receptor plays a pivotal role in regulating food intake and energy expenditure, and obesity results from mutations that interfere with the MC4 receptor pathway. We investigated the effect of glucocorticoids on endogenous MC4 receptors expressed in GT1-1 cells, an immortalized hypothalamic neuronal cell line. Dexamethasone (Dex) caused a 5- to 10-fold increase in the cAMP response to the MC4 receptor agonist, NDP-alphaMSH. The stimulatory effect of Dex reached a maximum within 24 h and was blocked by the glucocorticoid antagonist RU486. This glucocorticoid effect was specific for the MC4 receptor and not a result of up-regulation of another component of the cAMP cascade, because the response to endogenous beta-adrenergic receptor stimulation was not altered by Dex. Dex also potentiated NDP-alphaMSH-mediated ERK1/2 activation. After 12 h, Dex caused a 3- to 5-fold increase in [125I]NDP-alphaMSH binding, which was maintained for at least 48 h and prevented by RU486. Dex withdrawal caused a rapid return of MC4 receptor concentration to the basal level. Dex-mediated increases in MC4 receptor concentration resulted from a rapid but transient increase in MC4 receptor mRNA. This regulation apparently requires genomic regulatory sequences because Dex did not increase MC4 receptor expression or signaling in CHO cells expressing the MC4 receptor under the control of a cytomegalovirus promoter. We conclude that in GT1-1 hypothalamic neurons, glucocorticoids increase the amplitude of MC4 receptor signaling. This regulation may serve as a control to limit the effects of glucocorticoids on food intake.

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.

To investigate the function of dimerization of the TRH receptor, a controlled dimerization system was developed. A variant FK506 binding protein (FKBP) domain was fused to the receptor C terminus and dimerization induced by incubating cells with dimeric FKBP ligand, AP20187. The TRH receptor-fusion bound hormone and signaled normally. Addition of dimerizer to cells expressing the receptor-FKBP fusion dramatically increased the fraction of receptor running as dimer on SDS-PAGE. AP20187 caused dimerization in a time- and concentration-dependent manner, acting within 1 min. Dimerizer had no effect on TRH receptors lacking the FKBP domain, and its effects were blocked by excess monomeric FKBP ligand. AP20187-induced dimerization did not cause receptor phosphorylation, inositol phosphate production, or ERK1/2 activation, and dimerizer did not alter signaling by TRH. Induced dimerization did, however, alter TRH receptor trafficking. TRH promoted greater receptor internalization in cells treated with AP20187 but not monomeric ligand, based on loss of surface binding sites and immunostaining. Dimerization increased the rate of internalization of TRH receptors and decreased the apparent rate of receptor recycling. AP20187 enhanced the small amount of TRH-induced receptor internalization when the receptor-FKBP fusion protein was expressed in cells lacking beta-arrestins. The results show that controlled dimerization of the TRH receptor potentiates hormone-induced receptor trafficking.

Trafficking of TRH receptors was studied in a stable HEK293 cell line expressing receptor fused to a Timer protein (TRHR-Timer) that spontaneously changes from green to red over 10 h. Cells expressing TRHR-Timer responded to TRH with an 11-fold increase in inositol phosphate formation, increased intracellular free calcium, and internalization of 75% of bound [(3)H][N(3)-methyl-His(2)]TRH within 10 min. After a 20-min exposure to TRH at 37 C, 75-80% of surface binding sites disappeared as receptors internalized. When TRH was removed and cells incubated in hormone-free medium, approximately 75% of [(3)H][N(3)-methyl-His(2)]TRH binding sites reappeared at the surface over the next 2 h with or without cycloheximide. Trafficking of TRHR-Timer was monitored microscopically after addition and withdrawal of TRH. In untreated cells, both new (green) and old (red) receptors were seen at the plasma membrane, and TRH caused rapid movement of young and old receptors into cytoplasmic vesicles. When TRH was withdrawn, some TRHR-Timer reappeared at the plasma membrane after several hours, but much of the internalized receptor remained intracellular in vesicles that condensed to larger structures in perinuclear regions deeper within the cell. Strikingly, receptors that moved to the plasma membrane were generally younger (more green) than those that underwent endocytosis. There was no change in the red to green ratio over the course of the experiment in cells exposed to vehicle. The results indicate that, after agonist-driven receptor internalization, the plasma membrane is replenished with younger receptors, arising either from an intracellular pool or preferential recycling of younger receptors.

To study the effect of agonist on the TRH (thyrotrophin-releasing hormone) receptor protein, an epitope-tagged receptor was stably expressed in HEK-293 cells (human embryonic kidney 293 cells) and receptor levels were measured by immunoblotting. TRH caused a 5-25-fold increase in receptor protein during 48 h, which was half-maximal at 1 nM and was slowly reversible after hormone withdrawal. Chlordiazepoxide, an inverse agonist, had no effect. TRH up-regulation was mimicked by phorbol ester and blocked by the protein kinase C inhibitor GF109203X in combination with thapsigargin, which prevents a calcium response. TRH and phorbol ester increased the density of immunoreactive receptors localized at the cell surface and [3H]MeTRH (where MeTRH stands for [N3-methyl-His]TRH) binding. TRH also increased the concentration of a truncated, internalization-defective receptor. Analysis of cell lines stably expressing TRH receptors fused to the green fluorescent protein on a fluorescence-activated cell sorter showed that TRH and phorbol ester caused 2.7- and 6.8-fold increases in fusion protein expression respectively. TRH receptor up-regulation was only partially accounted for by changes in receptor mRNA, which increased 1.7-fold. TRH caused a small increase in receptor concentration in the presence of cycloheximide, actinomycin D or MG132. In contrast with the results obtained with the TRH receptor, agonist decreased the concentration of stably expressed b2-adrenergic receptors. These results show that TRH increases receptor concentration by a complex mechanism that requires signal transduction but not receptor endocytosis.

G protein-coupled receptors (GPCRs) are activated by a wide array of signals, which include light, neurotransmitters, hormones, cytokines, and drugs. Knowledge of the subcellular distribution of GPCRs is required in many experimental situations. Most GPCR signaling occurs in response to activators that interact with receptors localized on the cell surface. GPCRs must move from their site of synthesis to the plasma membrane, often undergoing redistribution in response to ligand binding. Endocytosis and recycling of receptors are important for desensitization and resensitization. Furthermore, mutations in GPCRs can alter receptor trafficking and prevent normal receptor function. This chapter describes a widely applicable method for visualizing a GPCR by indirect immunofluorescence microscopy.

These studies were designed to characterize ubiquitination of the G protein-coupled TRH receptor (TRHR). TRHRs and ubiquitin coprecipitated with antibodies to either receptor or ubiquitin in Chinese hamster ovary or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRHRs and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRHR coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRHRs were particularly heavily ubiquitinated. These results show that the proteasome participates in TRHR quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRHRs were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and nonpermissive temperatures, indicating that ligand-dependent ubiquitination of the receptor, or any other protein, is not necessary for TRHR signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.

The role of the Ca(2+)-induced Ca(2+) release channel (ryanodine receptor) in MIN6 pancreatic beta-cells was investigated. An endoplasmic reticulum (ER)-targeted "cameleon" was used to report lumenal free Ca(2+). Depolarization of MIN6 cells with KCl led to release of Ca(2+) from the ER. This ER Ca(2+) release was mimicked by treatment with the ryanodine receptor agonists caffeine and 4-chloro-m-cresol, reversed by voltage-gated Ca(2+) channel antagonists and blocked by treatment with antagonistic concentrations of ryanodine. The depolarization-induced rise in cytoplasmic Ca(2+) was also inhibited by ryanodine, which did not alter voltage-gated Ca(2+) channel activation. Both ER and cytoplasmic Ca(2+) changes induced by depolarization occurred in a dose-dependent manner. Glucose caused a delayed rise in cytoplasmic Ca(2+) but no detectable change in ER Ca(2+). Carbamyl choline caused ER Ca(2+) release, a response that was not altered by ryanodine. Taken together, these results provide strong evidence that Ca(2+)-induced Ca(2+) release augments cytoplasmic Ca(2+) signals in pancreatic beta-cells.

We previously developed peptides that bind to G protein betagamma subunits and selectively block interactions between betagamma subunits and a subset of effectors in vitro (Scott, J. K., Huang, S. F., Gangadhar, B. P., Samoriski, G. M., Clapp, P., Gross, R. A., Taussig, R., and Smrcka, A. V. (2001) EMBO J. 20, 767-776). Here, we created cell-permeating versions of some of these peptides by N-terminal modification with either myristate or the cell permeation sequence from human immunodeficiency virus TAT protein. The myristoylated betagamma-binding peptide (mSIRK) applied to primary rat arterial smooth muscle cells caused rapid activation of extracellular signal-regulated kinase 1/2 in the absence of an agonist. This activation did not occur if the peptide lacked a myristate at the N terminus, if the peptide had a single point mutation to eliminate betagamma subunit binding, or if the cells stably expressed the C terminus of betaARK1. A human immunodeficiency virus TAT-modified peptide (TAT-SIRK) and a myristoylated version of a second peptide (mSCAR) that binds to the same site on betagamma subunits as mSIRK, also caused extracellular signal-regulated kinase activation. mSIRK also stimulated Jun N-terminal kinase phosphorylation, p38 mitogen-activated protein kinase phosphorylation, and phospholipase C activity and caused Ca2+ release from internal stores. When tested with purified G protein subunits in vitro, SIRK promoted alpha subunit dissociation from betagamma subunits without stimulating nucleotide exchange. These data suggest a novel mechanism by which selective betagamma-binding peptides can release G protein betagamma subunits from heterotrimers to stimulate G protein pathways in cells.

Aim: Deletion of amino acids 32-71 in human growth hormone (Delta 32-71-GH) causes severe autosomal dominant GH deficiency. These experiments test whether retention of Delta 32-71-GH in the endoplasmic reticulum (ER) lumen leads to aberrant Ca(2+) regulation., Design: COS cells were transfected with Delta 32-71-GH, wild-type-GH or empty plasmid, and the ability of the cells to release Ca(2+) from the ER and transmit a Ca(2+) signal to the cytoplasm was investigated using cytoplasmic Ca(2+) dyes and ER-targeted cameleon Ca(2+) reporters., Results: Resting free Ca(2+), the rate of Ca(2+) release from the ER and the size of the ionophore-releasable ER Ca(2+) pool were not altered by Delta 32-71-GH. Stimulation of endogenous Ca(2+)-mobilizing receptors for histamine and thrombin resulted in similar changes in cytoplasmic and ER Ca(2+) in cells expressing wild-type and Delta 32-71-GH., Conclusion: Ca(2+) regulation is preserved despite retention of misfolded GH in the ER.

Dimerization and phosphorylation of thyrotropin-releasing hormone (TRH) receptors was characterized using HEK293 and pituitary GHFT cells expressing epitope-tagged receptors. TRH receptors tagged with FLAG and hemagglutinin epitopes were co-precipitated only if they were co-expressed, and 10-30% of receptors were isolated as hemagglutinin/FLAG-receptor dimers under basal conditions. The abundance of receptor dimers was increased when cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a dose-dependent manner. TRH increased phosphorylation of both receptor monomers and dimers, documented by incorporation of (32)P and an upshift in receptor mobility reversed by phosphatase treatment. The ability of TRH to increase receptor phosphorylation and dimerization did not depend on signal transduction, because it was not inhibited by the phospholipase C inhibitor. Receptor phosphorylation required an agonist but was not blocked by the casein kinase II inhibitor apigenin, the protein kinase C inhibitor GF109203X, or expression of a dominant negative form of G protein-coupled receptor kinase 2. TRH receptors lacking most of the cytoplasmic carboxyl terminus formed dimers constitutively but failed to undergo agonist-induced dimerization and phosphorylation. TRH also increased phosphorylation and dimerization of TRH receptors expressed in GHFT pre-lactotroph cells.

A large family of TRH-like peptides in the limbic region of rat brain including pGlu-Glu-Pro-NH(2) (EEP), pGlu-Val-Pro-NH(2) (Val(2)-TRH), Leu(2)-TRH, Phe(2)-TRH and Tyr(2)-TRH has recently been discovered. TRH (pGlu-His-Pro-NH(2)) has antidepressant, neuroprotective, analeptic, anticonvulsant, antiamnesic and euphoric properties, and other TRH-like peptides such as EEP exert several of these effects. A new TRH receptor (TRHR2) has been reported which is highly expressed in regions of rat brain that regulate attention and learning, arousal, sleep and processing of sensory information. The TRHR1 predominates in limbic structures involved in regulation of mood and in pituitary. This study examined the possibility that some of the newly discovered TRH-like peptides bind with high affinity to TRHR2, and that this receptor acts as the transducer for some of the CNS effects of this new class of neuropeptides. EEP, Val(2)-TRH and Leu(2)-TRH were analeptics, like TRH, but Phe(2)-TRH and Tyr(2)-TRH were not. The affinity and efficacy of TRH-like peptides for TRHR1 and TRHR2 were measured in HEK293 cells stably expressing these receptors. The IC(50) values of TRH-like peptides for displacement of [3H]TRH from TRHR2 were TRH<<<(Leu(2)-, Phe(2)-TRH)<(Gln(2)-, Ser(2)-TRH)<<(Val(2)-, Tyr(2)-, Arg(2)-, Thr(2)-, and Glu(2)-TRH). The IC(50) for Leu(2)-TRH was about 100 times that for TRH. When tested at the calculated IC(50) values, TRH-like peptides stimulated calcium responses in cells expressing TRHR1 and TRHR2, indicating that the peptides act as weak agonists at both receptors. These results indicate that TRHR1 and TRHR2 do not mediate the behavioral effects of TRH-like peptides.

In some individuals with autosomal dominant isolated growth hormone deficiency, one copy of growth hormone lacks amino acids 32-71 and is severely misfolded. We transfected COS7 cells with either wild-type human growth hormone or Delta 32-71 growth hormone and investigated subcellular localization of growth hormone and other proteins. Delta 32-71 growth hormone was retained in the endoplasmic reticulum, whereas wild-type hormone accumulated in the Golgi apparatus. When cells transfected with wild-type or Delta 32-71 growth hormone were dually stained for growth hormone and the Golgi markers beta-COP, membrin or 58K, wild-type growth hormone was colocalized with the Golgi markers, but beta-COP, membrin and 58K immunoreactivity was highly dispersed or undetectable in cells expressing Delta 32-71 growth hormone. Examination of alpha-tubulin immunostaining showed that the cytoplasmic microtubular arrangement was normal in cells expressing wild-type growth hormone, but microtubule-organizing centers were absent in nearly all cells expressing Delta 32-71 growth hormone. To determine whether Delta 32-71 growth hormone would alter trafficking of a plasma membrane protein, we cotransfected the cells with the thyrotropin-releasing hormone (TRH) receptor and either wild-type or Delta 32-71 growth hormone. Cells expressing Delta 32-71 growth hormone, unlike those expressing wild-type growth hormone, failed to show normal TRH receptor localization or binding. Expression of Delta 32-71 growth hormone also disrupted the trafficking of two secretory proteins, prolactin and secreted alkaline phosphatase. Delta 32-71 growth hormone only weakly elicited the unfolded protein response as indicated by induction of BiP mRNA. Pharmacological induction of the unfolded protein response partially prevented deletion mutant-induced Golgi fragmentation and partially restored normal TRH receptor trafficking. The ability of some misfolded proteins to block endoplasmic reticulum-to-Golgi traffic may explain their toxic effects on host cells and suggests possible strategies for therapeutic interventions.

To determine whether the interaction of the TRH receptor with beta-arrestin is necessary for TRH activation of MAPK, cells expressing either intact or truncated, internalization-defective TRH receptors were transfected with a beta-arrestin-green fluorescent protein conjugate. In cells expressing the wild-type pituitary TRH receptor, TRH caused translocation of the beta-arrestin-green fluorescent protein conjugate from the cytosol to the plasma membrane within 30 sec. After 5 min, the beta-arrestin-green fluorescent protein conjugate was visible in vesicles, where it colocalized with rhodamine-labeled TRH. In hypertonic sucrose, the beta-arrestin-green fluorescent protein conjugate translocated to the plasma membrane after TRH addition but did not internalize. In cells expressing the truncated TRH receptor, TRH did not cause translocation of the beta-arrestin-green fluorescent protein conjugate. TRH activated MAPK strongly in cells expressing intact or truncated TRH receptors, indicating that the receptor does not need to bind beta-arrestin or internalize. MAPK activation by TRH, epidermal growth factor, and phorbol ester was strongly inhibited by hypertonic sucrose and concanavalin A, which block movement of proteins into coated pits and coated pit assembly. Hypertonic sucrose did not affect MAPK activation in cells overexpressing MAPK kinase 1. Dominant negative dynamin, which blocks conversion of coated pits to vesicles, also reduced receptor internalization and TRH activation of MAPK. TRH activation of MAPK required PKC but was insensitive to pertussis toxin and did not require ras, epidermal growth factor receptor kinase, or PI3K. These results show that the TRH receptor itself does not need to bind beta-arrestin or undergo sequestration to activate MAPK but that the endocytic pathway must be intact.

Burkitt's lymphoma cell lines have been important in vitro models for studying the pathogenesis of Burkitt's lymphoma (BL) and for exploring new treatment strategies. A new EBV(-) Burkitt's lymphoma cell line (GA-10) was established from a patient with a clinically aggressive, chemorefractory BL and characterized. Although functional p-glycoprotein could not be demonstrated by dye-efflux assays, both p53 genes were mutated in the GA-10 cells, perhaps contributing to the resistant phenotype of the original neoplasm. Two properties of BL cells which may be useful targets for novel cytotoxic therapeutics are their surface expression of CD77, the receptor for Shiga toxin (Stx), and their high rate of proliferation. Expression of CD77 on the GA-10 cells was heterogeneous in that certain subclones expressed high levels of CD77 and correspondingly exhibited strong growth inhibition by Stx while others showed low levels of CD77 expression and weak Stx-induced growth inhibition. Flavopiridol, a potent inhibitor of cell cycle progression through G1 and G2, induced cytotoxicity of the GA-10 cells with an LC(50) of approximately 40 nM vs 70 nM for HL-60 cells (P < 0.05). The concentrations of flavopiridol at which only 10% of the cells were viable (LC(10)) were approximately 280 nM for the GA-10 cells and 520 nM for the HL-60 cells (P < 0.05). Dose-related induction of apoptosis in response to flavopiridol was demonstrated in the GA-10 cells by morphology, TUNEL assay, and activation of caspase-3. Flavopiridol was also cytotoxic to seven other BL cell lines tested. These data suggest that flavopiridol may have therapeutic value in the treatment of Burkitt's lymphoma., (Copyright 2001 Academic Press.)

These studies examined the importance of phospholipase Cbeta (PLCbeta) in the calcium responses of pituitary cells using PLCbeta3 knockout mice. Pituitary tissue from wild-type mice contained PLCbeta1 and PLCbeta3 but not PLCbeta2 or PLCbeta4. Both Galphaq/11 and Gbetagamma can activate PLCbeta3, whereas only Galphaq/11 activates PLCss1 effectively. In knockout mice, PLCbeta3 was absent, PLCbeta1 was not up-regulated, and PLCbeta2 and PLCbeta4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCbeta3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCbeta3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCbeta3 knockout animals These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCbeta3 through Gbetagamma, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCbeta1 and PLCbeta3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 microM TRH and GnRH were identical in pituitary cells from wild-type and PLCbeta3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCbeta1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCbeta3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.

HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca(2+) indicators designed to measure the free Ca(2+) concentration in the cytoplasm, [Ca(2+)](cyt), and the endoplasmic reticulum (ER), [Ca(2+)](er). Basal [Ca(2+)](cyt) was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca(2+)](cyt) to 1 micrometer or higher. Basal [Ca(2+)](er) averaged 500 micrometer and fell to 50-100 micrometer over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca(2+)](er) to 100 micrometer, independent of extracellular Ca(2+), whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca(2+)](er) to 5-10 micrometer, indicating that there is substantial store refilling during signaling. TRH increased [Ca(2+)](cyt) and decreased [Ca(2+)](er) if applied after other agonists, whereas other agonists did not alter [Ca(2+)](cyt) or [Ca(2+)](er) if added after TRH. When Ca(2+) was added back to cells that had been incubated with TRH in Ca(2+)-free medium, [Ca(2+)](cyt) and [Ca(2+)](er) increased rapidly. The increase in [Ca(2+)](er) was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In conclusion, these new Ca(2+) indicators showed that basal [Ca(2+)](er) is approximately 500 micrometer, that [Ca(2+)](er) has to be >100 micrometer to support an increase in [Ca(2+)](cyt) by agonists, and that during signaling, intracellular Ca(2+) stores are continuously refilled with cytoplasmic Ca(2+) by the sarcoendoplasmic reticulum Ca(2+)-ATPase pump.

Morphine and other micro opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC beta3-deficient mice, we have established a strong link between PLC and mu opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC beta3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED(50) value for morphine in producing antinociception. The reduced ED(50) value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B(max) and K(d) values for mu, kappa, and delta opioid receptors between wild-type and PLC beta3-null mice. We also found that opioid regulation of voltage-sensitive Ca(2+) channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific mu agonist [D-Ala(2),(Me)Phe(4),Gly(ol)(5)]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC beta3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC beta3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca(2+) current reduction. These data demonstrate that PLC beta3 constitutes a significant pathway involved in negative modulation of mu opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.