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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 [3H]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.

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.

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.

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. This work was supported in part by the grants from Research funding of the Madrid Autonomous Region ENDOSCREEN S2010/BMD-2396 (to J.C.M.) and the European Regional Development Fund (ERDF-FEDER) and the Spanish Ministry of Economy and Competitiveness SAF2010-19230 (to M.P.M.); and from the Social Funds of the European Community (ERDF-FEDER) and the Spanish Ministry of Economy and Competitiveness BFU2013-46109-R (to C.V.A.), from the Social Funds of the European Community (ERDF-FEDER) and the Xunta de Galicia R2014/050 REDICENT (to C.V.A.); and from Instituto de Salud Carlos III PI15/01501- FEDER (to J.M.C-T.) SI

Accessory to Obesity? Melanocortin receptors are a family of cell membrane receptors that control diverse physiological functions. Mutations in the gene encoding melanocortin 4 receptor (MC4R) are a cause of familial early-onset obesity. Asai et al. (p. 275 ) studied the function of an accessory protein for MC4R signaling, MRAP2, and found that mice genetically deficient in MRAP2 develop severe obesity. Sequencing of MRAP2 in unrelated, severely obese humans revealed one individual with a clearly disruptive genetic variant, suggesting that MRAP2 mutations might also be a rare cause of human obesity. In a zebrafish model, Sebag et al. (p. 278 ) studied two paralogs of the MRAP2 accessory protein, one of which enhanced MC4R responsiveness to α–melanocyte-stimulating hormone, which regulates feeding and growth.

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.

The melanocortin2 (MC21), 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.

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.

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.

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.

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 [3H][N3-methyl-His2]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 [3H][N3-methyl-His2]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.

The role of the Ca2+-induced Ca2+ release channel (ryanodine receptor) in MIN6 pancreatic β-cells was investigated. An endoplasmic reticulum (ER)-targeted “cameleon” was used to report lumenal free Ca2+. Depolarization of MIN6 cells with KCl led to release of Ca2+ from the ER. This ER Ca2+ release was mimicked by treatment with the ryanodine receptor agonists caffeine and 4-chloro-m-cresol, reversed by voltage-gated Ca2+ channel antagonists and blocked by treatment with antagonistic concentrations of ryanodine. The depolarization-induced rise in cytoplasmic Ca2+ was also inhibited by ryanodine, which did not alter voltage-gated Ca2+ channel activation. Both ER and cytoplasmic Ca2+ changes induced by depolarization occurred in a dose-dependent manner. Glucose caused a delayed rise in cytoplasmic Ca2+ but no detectable change in ER Ca2+. Carbamyl choline caused ER Ca2+ release, a response that was not altered by ryanodine. Taken together, these results provide strong evidence that Ca2+-induced Ca2+ release augments cytoplasmic Ca2+ signals in pancreatic β-cells.

Aim : Deletion of amino acids 32–71 in human growth hormone (Δ32–71-GH) causes severe autosomal dominant GH deficiency. These experiments test whether retention of Δ32–71-GH in the endoplasmic reticulum (ER) lumen leads to aberrant Ca 2+ regulation. Design : COS cells were transfected with Δ32–71-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 Δ32–71-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 Δ32–71-GH. Conclusion : Ca 2+ regulation is preserved despite retention of misfolded GH in the ER.

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 [ 3 H]TRH from TRHR2 were TRH⋘(Leu 2 -, Phe 2 -TRH) 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.

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.

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 Ca2+ indicators designed to measure the free Ca2+ concentration in the cytoplasm, [Ca2+]cyt, and the endoplasmic reticulum (ER), [Ca2+]er. Basal [Ca2+]cyt was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca2+]cyt to 1 μm or higher. Basal [Ca2+]er averaged 500 μmand fell to 50–100 μm over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca2+]er to 100 μm, independent of extracellular Ca2+, whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca2+]er to 5–10 μm, indicating that there is substantial store refilling during signaling. TRH increased [Ca2+]cyt and decreased [Ca2+]er if applied after other agonists, whereas other agonists did not alter [Ca2+]cyt or [Ca2+]er if added after TRH. When Ca2+ was added back to cells that had been incubated with TRH in Ca2+-free medium, [Ca2+]cyt and [Ca2+]er increased rapidly. The increase in [Ca2+]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 Ca2+ indicators showed that basal [Ca2+]er is ∼500 μm, that [Ca2+]er has to be >100 μm to support an increase in [Ca2+]cyt by agonists, and that during signaling, intracellular Ca2+ stores are continuously refilled with cytoplasmic Ca2+ by the sarcoendoplasmic reticulum Ca2+-ATPase pump.

The thyrotropin-releasing hormone (TRH) receptor was expressed in embryonic fibroblasts from mice lacking the alpha subunits of Gq and G11 (Fq/11 cells) to determine whether G protein coupling is necessary for agonist-dependent receptor internalization. Neither TRH nor agonists acting on endogenous receptors increased intracellular calcium unless the cells were co-transfected with the alpha subunit of Gq. In contrast, temperature-dependent internalization of [3H]MeTRH in Fq/11 cells was the same whether Gqalpha was expressed or not. A rhodamine-labeled TRH analog and fluorescein-labeled transferrin co-localized in endocytic vesicles in Fq/11 cells, indicating that endocytosis took place via the normal clathrin pathway. Cotransfection with beta-arrestin or V53D beta-arrestin increased TRH-dependent receptor sequestration. Fq/11 cells were co-transfected with the TRH receptor and a green fluorescent protein (GFP)-beta-arrestin conjugate. GFP-beta-arrestin was uniformly distributed in the cytoplasm of untreated cells and quickly translocated to the periphery of the cells when TRH was added. A truncated TRH receptor that lacks potential phosphorylation sites in the cytoplasmic carboxyl terminus signaled but did not internalize or cause membrane localization of GFP-beta-arrestin. These results prove that calcium signaling by the TRH receptor requires coupling to a G protein in the Gq family, but TRH-dependent binding of beta-arrestin and sequestration do not.

Previous studies have implied that a transcription factor(s) other than Pit-1 is involved in homeostatic regulation of PRL promoter activity via Pit-1-binding elements. One such element, 1P, was employed to clone from a rat pituitary cDNA expression library a novel 417-amino acid WD protein, designated PREB (PRL regulatory element binding) protein. PREB contains two PQ-rich potential transactivation domains, but no apparent DNA-binding motif, and exhibits sequence-specific binding to site 1P, to a site nonidentical to that for Pit-1. The PREB gene (or a related gene) is conserved, as an apparently single copy, in rat, human, fly, and yeast. A single approximately 1.9-kb PREB transcript accumulates in GH3 rat pituitary cells, to levels similar to Pit-1 mRNA. PREB transcripts were detected in all human tissues examined, but the observation of tissue-specific multiple transcript patterns suggests the possibility of tissue-specific alternative splicing. RT-PCR analysis of human brain tumor RNA samples suggested region-specific expression of PREB transcripts in brain. Western and immunocytochemical analysis implied that PREB accumulates specifically in GH3 cell nuclei. Transient transfection employing PREB-negative C6 rat glial cells showed that PREB is as active as, and additive with, Pit-1 in transactivation of a PRL promoter construct, and that PREB, but not Pit-1, can mediate transcriptional activation by protein kinase A (PKA). Expression in GH3 cells of a GAL4-PREB fusion protein both strongly transactivated a 5XGAL indicator construct and yielded a further stimulation of expression of this construct by coexpressed PKA, implying that PREB can mediate both basal and PKA-stimulated transcriptional responses in pituitary cells. These observations imply that PREB will prove to play a significant transcriptional regulatory role, both in the pituitary and in other organs in which transcripts of its gene are expressed.

Primary cultures of rat pituitary cells were stained with an antibody to the native thyrotropin-releasing hormone (TRH) receptor and with a bioactive, fluorescent analogue of TRH, Rhod-TRH. Rhod-TRH specifically stained 86% of lactotropes and 21% of nonlactotropes from primary pituitary cell cultures. Lactotropes and thyrotropes accounted for 90% of cells that stained with Rhod-TRH, but there were occasional lactotropes and thyrotropes that did not show detectable staining with antireceptor antibodies or with Rhod-TRH. The intensity of staining was generally higher in the GH3 line of tumor cells than in normal pituicytes, and 100% of the tumor cells stained with Rhod-TRH. To determine whether the TRH receptor undergoes ligand-directed endocytosis in normal cells, TRH receptor immunocytochemistry was performed before and after TRH binding. TRH receptors were localized on the surface of cells prior to TRH exposure, and Rhod-TRH fluorescence was confined to the plasma membrane when TRH binding was performed at 0 degrees C, where endocytosis is blocked. When cells were incubated with TRH at 37 degrees C, receptors were found in intracellular vesicles in both lactotropes and thyrotropes, and Rhod-TRH was rapidly internalized into endosomes at elevated temperatures. Internalization of Rhod-TRH was inhibited by hypertonic sucrose, indicating that it occurs through clathrin-coated pits. These findings show that some of the heterogeneity in the secretory and calcium responses of pituicytes to TRH occurs at the level of the TRH receptor.

Purpose: Microscopic methods to measure the activity of drug extrusion systems important in multidrug resistance in individual cells were developed. Methods: Multidrug-resistant (MDR) and parental lines of hamster CHO and pituitary GH3 cells were incubated with the acetoxymethylester (AM) forms of several fluorescent calcium-sensing dyes, fura2, indo1 and fluo3. The AM forms of these compounds are hydrolyzed by intracellular esterases and then trapped in cells, and the AM forms of the dyes are excellent substrates for P-glycoprotein (Pgp). Results: The fluorescent free acid forms of fura2, indo1 and fluo3 did not accumulate in MDR lines unless a chemosensitizer such as cyclosporin A, R(+)verapamil, quinidine, or progesterone was included during loading to prevent the cells from extruding the AM forms of the dyes before they could be hydrolyzed. Cyclosporin A increased the fluorescence due to intracellularly trapped fura2 free acid from 8- to 20-fold and was maximally effective at

Three independent methods were used to block internalization of the TRH receptor: cells were infected with vaccinia virus encoding a dominant negative dynamin, incubated in hypertonic sucrose, or stably transfected with a receptor lacking the C-terminal tail. Internalization was blocked in all three paradigms as judged by microscopy using a fluorescently labeled TRH agonist and biochemically. The initial inositol trisphosphate (IP3) and Ca2+ responses to TRH were normal when internalization was inhibited. The IP3 increase was sustained rather than transient, however, in cells expressing the truncated TRH receptor, implying that the C-terminal tail of the receptor may be important for uncoupling from phospholipase C. After withdrawal of TRH, cells were refractory to TRH until both ligand dissociation and resensitization of the receptor had occurred. When surface-bound TRH was removed by a mild acid wash, which did not impair receptor function, neither wild-type nor truncated receptors were able to generate full IP3 responses for about 10 min. The rate of recovery was not altered by blocking internalization. Recovery of intracellular Ca2+ responses also depended on the rate of Ca2+ pool refilling. In summary, in the continued presence of TRH, phospholipase C activity declines quickly due to receptor uncoupling; this desensitization does not take place for the truncated receptor. After TRH is withdrawn, cells are refractory to TRH. Before cells can respond, TRH must dissociate and a resensitization step, which takes place on the plasma membrane and does not require the C-terminal tail of the receptor, must occur.

The mechanism by which lead crosses the blood-brain barrier is not known. Brain capillary endothelial cells, which form tight junctions with each other, are an important component of the blood-brain barrier. Lead must traverse these cells to reach the brain. In the present study, uptake of lead was followed in primary cultures of bovine brain capillary endothelial cells. Lead uptake into cells was measured by monitoring the fluorescence of cells loaded with indo-1 at a wavelength where indo-1 fluorescence is independent of calcium but quenched by binding of lead. Lead uptake was visualized with digital images recorded with a fluorescence microscope. Lead added to the extracellular medium caused fluorescence quench over time which was reversed upon addition of a membrane permeant heavy metal chelator. Lead uptake by cells in suspension, measured by fluorescence spectroscopy, exhibited time and concentration dependence. Lead uptake was enhanced following depletion of intracellular Ca2+ stores by the addition of thapsigargin, cyclopiazonic acid, or tert-butylhydroquinone, inhibitors of the sarco/endoplasmic reticulum calcium ATPase. SK&F 96365, which blocks store-operated calcium channels, inhibited the stimulation of lead uptake by thapsigargin. These results indicate that indo-1 fluorescence quench is a useful method for investigation of lead uptake in brain capillary endothelial cells. Furthermore, entry of lead into these cells is activated by the depletion of intracellular Ca2+ stores and may occur via store-operated cation channels.

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.

Thyrotropin-releasing hormone (TRH) acts via a G-protein-coupled receptor on lactotrophs to increase the intracellular free calcium ion concentration, [Ca 2+ ] i . The [Ca 2+ ] i response depends on both TRH concentration and the duration of TRH exposure. An initial, short-lived [Ca 2+ ] i spike results from release of Ca 2+ from intracellular stores, whereas a later sustained [Ca 2+ ] i increase, often characterized by [Ca 2+ ] i oscillations, results from an influx of extracellular Ca 2+ through both voltage-gated and non-voltage-gated, store-operated Ca 2+ channels. The initial spike phase predominates at high doses of TRH, whereas the plateau phase predominates at low doses. The mechanisms underlying the complex [Ca 2+ ] i response to TRH are discussed.

TRH stimulates rat (r) TSH beta gene promoter activity at two distinct response elements, which also respond to protein kinase C-signaling pathways. The dependence of TRH-stimulated transcription of the TSH beta gene on a rise in intracellular calcium [Ca2+]i, and on the necessity for Ca2+ influx through L-type voltage-gated calcium channels was investigated in two transfected cell lines and in normal thyrotropes. The transcription rate of the homologous gene in normal thyrotropes was measured by nuclear run-off assays. Bay K8644, an L channel agonist, stimulated TSH beta gene transcription 6-fold, and TRH stimulation of TSH beta gene transcription was partially blocked by nimodipine, an L channel antagonist, while phorbol 12-myristate-13-acetate (PMA)-stimulated transcription was not. Bay K8644 plus TRH had a greater effect than either treatment alone. Constructs of the 5'-flanking region of the TSH beta gene fused to the luciferase reporter (TSH beta LUC) were then transfected into excitable GH3 pituitary cells. TSH beta LUC was stimulated 2- to 5-fold by 1 nM TRH or 100 nM Bay K8644, and the TRH effect was nearly abolished by nimodipine or chelation of external Ca2+. Constructs containing isolated TRH-responsive elements fused to a heterologous promoter responded similarly. The protein kinase C activator, PMA (100 nM) also stimulated TSH beta LUC transcription, but its effect was not inhibited by nimodipine. A stable heterologous cell line containing the mouse TRH receptor was constructed by transfection of nonexcitable 293 cells, which lack L channel activity. In the resultant 301 cells, TSH beta LUC activity was increased 2- to 3-fold by TRH or PMA; nimodipine, Bay K8644, and removal of extracellular Ca2+ had no effect. We conclude that TRH stimulation of TSH beta gene transcription requires Ca2+ release from inositol triphosphate-sensitive stores and Ca2+ influx via L-type calcium channels in GH3 cells, but in transfected 293 cells TRH activation of protein kinase C plays a predominant role in activating TSH beta. Both mechanisms appear to be operative in normal thyrotropes.

Endocytosis and recycling of both thyrotropin-releasing hormone (TRH) and its G-protein-coupled receptor were visualized by conventional and confocal fluorescence microscopy in pituitary cells using a rhodamine-labeled TRH analog (Rhod-TRH) and indirect immunofluorescent staining of cells stably transfected with an epitope-tagged TRH receptor (TRHR). The epitope-tagged TRHR was confined to the cell surface prior to agonist treatment. Both Rhod-TRH and TRHR were also localized on the plasma membrane after agonist binding at 0 degrees C. Ligand binding at 37 degrees C resulted in rapid endocytosis, and both Rhod-TRH and the epitope-tagged TRHR appeared in cytoplasmic vesicles within 5 min. Fluorescently labeled TRH and transferrin colocalized in the same endocytotic vesicles, and internalization of Rhod-TRH and TRHR was inhibited by hypertonic medium, suggesting that endocytosis occurred by a clathrin-dependent mechanism. Internalized TRHRs returned to the membrane within 20 min after removal of TRH, and cycloheximide did not block receptor recycling. A mutant TRHR truncated at Cys335 signaled but did not internalize Rhod-TRH, confirming the importance of the carboxyl terminus of the TRHR in receptor-mediated endocytosis. Thus, the TRH-TRHR complex is endocytosed via clathrin-coated vesicles and the receptor is recycled to the plasma membrane.

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 minutes 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 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.

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.

These studies characterize the mechanisms involved in terminating the initial Ca2+ transient stimulated by thyrotropin-releasing hormone (TRH). When TRH was added to GH3 pituitary cells that had been treated with thapsigargin to block any agonist-stimulated increase in [Ca2+]i, TRH caused a decrease in [Ca2+]i. The Ca2+ clearing response was seen in pituitary GH3 cells and in nonexcitable HEK 293 cells transfected with TRH receptor cDNA, was evident at basal or elevated [Ca2+]i, and was mediated by the TRH receptor. The Ca2+ clearing response to TRH was not prevented by thapsigargin, Ca2+ ionophores, nimodipine, or replacement of extracellular Na+ but was inhibited by La3+. La3+ also increased the duration of the TRH-evoked [Ca2+]i transient. TRH-stimulated Ca2+ extrusion was directly demonstrated using extracellular fluo-3 free acid. TRH produced a 5-20-fold increase in Ca2+ efflux that was independent of extracellular Na+ and inhibited by vanadate. TRH stimulation of Ca2+ efflux was not reproduced by phorbol esters or inhibited by down-regulation of protein kinase C or staurosporine. The results suggest that agonist-activated Ca2+ efflux may be a universal component of an agonist-activated Ca2+ response and further suggest that a plasma membrane Ca2+ pump may be an effector for G-protein-coupled receptors linked to Ca2+ mobilization.

The Ca2+ response of individual cells to TRH was investigated in excitable pituitary GH3 and in nonexcitable Hela cells transfected with the TRH receptor complementary DNA (HelaR cells). GH3 cells typically responded to 1 microM TRH with an immediate transient [Ca2+]i spike (mean peak [Ca2+]i = 1.5 microM) followed by a period of inactivity of approximately 100 sec long and then a secondary increase in [Ca2+]i with oscillations. At 10-100 nM TRH, the initial [Ca2+]i spike was more prolonged and immediately followed by a sustained elevation of [Ca2+]i. At 0.5-1 nM TRH, there was a variable lag before any response; the initial [Ca2+]i spike was absent or small, but the sustained phase was still present. The second phase of elevated [Ca2+]i, which could be eliminated with nimodipine or chelation of extracellular Ca2+, gave a bell-shaped TRH dose response curve. The effect of TRH on Ca2+ oscillations depended both on TRH concentration and the basal oscillation frequency. HelaR cells responded to 1 microM TRH with a rapid [Ca2+]i spike, and at less than or equal to 10 nM TRH, up to 50% of HelaR cells displayed agonist-induced sinusoidal [Ca2+]i oscillations independent of extracellular Ca2+. TRH never caused a sustained elevation of [Ca2+]i in HelaR cells. For GH3 and HelaR cells, the peak [Ca2+]i response increased with TRH concentration up to 1 microM. In contrast, the duration of the initial [Ca2+]i spike was shorter at higher TRH concentrations, decreasing from 16 to 6.3 s (GH3) or 92 to 35 s (HelaR) between 0.5 nM and 1 microM TRH. This shortening of the spike duration was caused by rapid clearing of cytoplasmic Ca2+ that depended primarily on agonist concentration. In summary, TRH stimulates a complex [Ca2+]i response pattern dependent upon both the agonist concentration and cell context. The initial burst of Ca2+ is cleared in part by agonist dependent Ca2+ clearing.

Thapsigargin (TG), 2,5-t-butylhydroquinone (tBHQ) and cyclopiazonic acid (CPA) all inhibit the initial Ca(2+)-response to thyrotropin-releasing hormone (TRH) by depleting intracellular Ca2+ pools sensitive to inositol 1,4,5-trisphosphate (IP3). Treatment of GH3 pituitary cells for 30 min with 5 nM TG, 500 nM tBHQ or 50 nM CPA completely eliminated the TRH-induced spike in intracellular free Ca2+ ([Ca2+]i). Higher concentrations of TG and tBHQ, but not CPA, were also found to inhibit strongly the activity of L-type calcium channels, as measured by the increase in [Ca2+]i or 45Ca2+ influx stimulated by depolarization. TG and tBHQ blocked high-K(+)-stimulated 45Ca2+ uptake, with IC50 values of 10 and 1 microM respectively. Maximal inhibition of L-channel activity was achieved 15-30 min after drug addition. Inhibition by tBHQ was reversible, whereas inhibition by TG was not. TG and CPA did not affect spontaneous [Ca2+]i oscillations when tested at concentrations adequate to deplete the IP3-sensitive Ca2+ pool. However, 20 microM TG and 10 microM tBHQ blocked [Ca2+]i oscillations completely. The effect of drugs on calcium currents was measured directly by using the patch-clamp technique. When added to the external bath, 10 microM CPA caused a sustained increase in the calcium-channel current amplitude over 8 min, 10 microM tBHQ caused a progressive inhibition, and 10 microM TG caused an enhancement followed by a sustained block of the calcium current over 8 min. In summary, CPA depletes IP3-sensitive Ca2+ stores and does not inhibit voltage-operated calcium channels. At sufficiently low concentrations, TG depletes IP3-sensitive stores without inhibiting L-channel activity, but, for tBHQ, inhibition of calcium channels occurs at concentrations close to those needed to block agonist mobilization of intracellular Ca2+.

We use an adenovirus vector, AdCMVmTRHR, to express thyrotropin-releasing hormone (TRH) receptors (TRH-Rs) to determine whether the size of the hormone-responsive phosphoinositide pool in mammalian cells is directly related to receptor number. Infection of HeLa cells with increasing numbers of AdCMVmTRHR caused time-dependent graded expression of TRH-Rs. Measurement of cytoplasmic free Ca2+ in individual cells permitted quantitation of the fraction of cells responsive to TRH. Infection with 100 AdCMVmTRHR particles/cell or more led to TRH responsiveness in > or = 90% of HeLa cells. Measurement of prelabeled phosphoinositides hydrolyzed during prolonged TRH stimulation assesses the size of the TRH-responsive pool. In cells infected with AdCMVmTRHR for 24 h, the size of the TRH-responsive phosphoinositide pool increased with increasing TRH-R expression. The TRH-responsive pool also increased with time after infection as the number of TRH-Rs increased. Similar observations were made in GHY and KB cells. These data confirm our previous suggestion (Cubitt, A. B., Geras-Raaka, E., and Gershengorn, M. C. (1990) Biochem. J. 271, 331-336) that there are hormone-responsive and -unresponsive pools of cellular phosphoinositides and that the maximal size of the TRH-responsive pool is directly related to the number of TRH-Rs.

The uptake and toxicity of cadmium were compared in two rat pheochromocytoma cell lines: PC12 cells, which express voltage-sensitive calcium channels, and PC18 cells, which do not. PC12 but not PC18 cells responded to depolarization with an increase in 45 Ca 2+ uptake and an increase in the concentration of cyzoplasmic free calcium ion, [Ca 2+ ] i . These responses were blocked by the dihydropyridine calcium channel antagonist nimodipine and amplified by the agonist BAY K8644, drugs selective for L-type channels. Cadmium caused death of PC12 cells with an LC5O of 12 μM. Inclusion of high K + with the agonist BAY K8644 shifted the kill curve to the left (LC5O = 6 μM), whereas nimodipine protected against cadmium toxicity (LC5O = 30 μM). In contrast, drugs acting on L-type calcium channels did not affect Cd 2+ toxicity for PC18 cells (LC5O 15 μM). Fura 2 was used to measure intracellular free Cd 2+ by fluorescence ratio methods. Addition of 25 pM Cd 2+ to both PC12 and PC18 cells caused a rise in the 340/380 fluorescence ratio attributable to the uptake of Cd 2+ , since it was almost completely reversed by chelating extracellular Cd 2+ and adding a membrane-permeant chelator of heavy metals. Cd 2+ addition resulted in a gradual increase in Fura 2 fluorescence in both PC12 and PC18 cells, but depolarization with BAY K8644 increased the apparent rate of Cd 2+ uptake only for the PC12 cells. Cd 2+ fluorescence appeared to be concentrated near the plasma membrane. The results confirm the potential involvement of calcium channels in cadmium transport and extend the use of intracellularly trapped fluorescent dyes to monitor intracellular free cadmium ion concentration.

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.

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.

The mechanism of uptake of the thyroid hormones, 3,5,3'-triiodo-L-thyronine (L-T3) and L-thyroxine (L-T4), was studied in rat pituitary GH4C1 cells. The major portion (approximately 65%) of L-T3 transport was stereospecific and saturable. Transport of L-T3 was 8-10 times more rapid than transport of D-T3. [125I]L-T3 transport was saturable at microM concentrations; a Lineweaver-Burk plot was linear with Km = 0.4 microM and Vmax = 4 pmol/min/10(6) cells. Unlabeled analogs competed with [125I]L-T3 uptake in the order L-T3 > or = L-T4 >> 3,3',5'-triiodo-L-thyronine (reverse-T3), D-T3, D-T4, and L-thyronine. L-T3 and L-T4 also both effectively inhibited [125I]L-T4 transport. Uptake of [125I]L-T3 was inhibited 40-55% by large neutral amino acids and 77% by 80 microM beta-2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, an inhibitor selective for the L system of amino acid uptake. Conversely, L-T3 inhibited the transport of [3H]leucine by pituitary cells (IC50 = 2 microM), but D-T3 and 3,5,3'-triiodothyroacetic acid (Triac) did not. L-Leucine was transported much more efficiently (Vmax = 0.65 mumol/min/10(6) cells) than L-T3 by GH4C1 cells. The results show that L-T3 and L-T4 share the same stereospecific transport pathway in pituitary cells, that the transport mechanism is saturable at supraphysiological thyroid hormone concentrations, and that the L system is partially responsible for L-T3 transport.

To determine the function of the E5 open reading frame (ORF) of the human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts were transformed to colony formation in soft agar, and the transformation frequency was increased by epidermal growth factor (EGF) but not by platelet-derived growth factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-16 were mitogenic in primary human foreskin epithelial cells (keratinocytes) and acted synergistically with EGF. Investigation of keratinocytes expressing HPV-16 E5 showed that the number of endogenous EGF receptors (EGFRs) per cell was increased two- to fivefold. Immunofluorescence microscopy of HPV-16 E5-expressing keratinocytes indicated that there was an apparent delay in the internalization and degradation of EGFRs compared with controls. Kinetic studies with [125I]EGF showed that the ligand underwent normal internalization and degradation in both HPV-16 E5-expressing and control keratinocytes, but in E5-expressing cells, a greater number of receptors recycled back to the cell surface within 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphorylation of the EGFR on tyrosine, an indication of receptor kinase activity, was of greater magnitude in the HPV-16 E5-expressing keratinocytes than in control cells, although the basal level of receptor phosphorylation was similar.

GH3 rat pituitary cells have L-type voltage-gated calcium channels, and both the rate of uptake of 45Ca2+ and the concentration of intracellular free calcium ion ([Ca2+]i) are increased by depolarization with high potassium. Cells incubated for several days with 10 nM epidermal growth factor (EGF) responded to depolarization with a 30-65% smaller increase in 45Ca2+ uptake than untreated cells. The inhibitory response to EGF developed slowly, with a maximal effect requiring 24-48 h. EGF exerted a half-maximal reduction in depolarization-stimulated 45Ca2+ uptake at 0.1 nM and a maximal effect at 1-10 nM. 45Ca2+ uptake was reduced by EGF at strongly depolarized potentials (added K+,25 mM) with or without the calcium channel agonist BAY K8644. [Ca2+]i was measured using fura-2 before and after depolarization in control cells and cells incubated for 48 h with 10 nM EGF. EGF-treated cells responded to the addition of 30-50 mM KCl with a smaller increase in [Ca2+]i than control cells. Digital fluorescence imaging of individual fura-2-loaded cells confirmed that the average [Ca2+]i response to depolarization was lower in cells that had been incubated with EGF for 36 h. EGF treatment increased the amount of PRL secreted basally, but inhibited the acute PRL secretory response to depolarization with 50 mM KCl and 1 microM BAY K8644 from 2.6- to 1.5-fold. The results indicate that EGF reduces the activity of voltage-gated dihydropyridine-sensitive calcium channels on pituitary cells, and that this reduction in L-channel activity is accompanied by a smaller secretory response to depolarization.

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.

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.

Cellular uptake of Cd2+ has been monitored using intracellularly trapped dyes, Fura 2 and Quin 2, which bind Cd2+ with extremely high affinity, and digital fluorescence imaging has been used to visualize intracellular free Cd2+. The excitation spectrum of the Cd2+ complex of Fura 2 is similar to that of the Ca2+ complex, whereas Cd2+ displaces Ca2+ from Quin 2 and reduces fluorescence. Fluorescence of Fura 2-loaded cells increased when 50 microM extracellular Cd2+ was added and fluorescence of Quin 2-loaded cells decreased. Cd2+ uptake by GH3 pituitary cells, which occurs in part via voltage-sensitive L-type calcium channels, was increased by BAY K8644 and depolarization and decreased by nimodipine. When Fura 2 and Quin 2 were used to measure Cd2+ uptake by glial C6 cells, which have no L-channel activity, high K+ and BAY K8644 did not change the apparent rate of Cd2+ uptake. GH3 and C6 cells were incubated with Cd2+ for 24 h and loaded with Fura 2, and fluorescence was measured before and after addition of tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), a membrane permeant chelator with extremely high affinity for metals. TPEN had little effect on fluorescence of Fura 2-loaded GH3 and C6 cells not exposed to Cd2+ but decreased fluorescence of cells that had been incubated with 1-10 microM Cd2+. Fluorescence ratio imaging of Fura 2-loaded cells was used to image intracellular free Cd2+ for both GH3 and C6 cells. Cd2+ uptake over 30-180 min could be followed by the increase in 340/380 fluorescence ratio and the increase in fluorescence ratio was reversed within 5 min by TPEN. The results provide further evidence for the importance of voltage-gated calcium channels to Cd2+ uptake of certain cells.

Hepatic microsomes and isolated hepatocytes in short term culture desulfate T3 sulfate (T3SO4). We, therefore, wished to determine whether T3SO4 could mimic the action of thyroid hormone in vitro. T3SO4 had no thyromimetic effect on the activity of Ca(2+)-ATPase in human erythrocyte membranes at doses up to 10,000 times the maximally effective dose of T3 (10(-10) mol/L). In GH4C1 pituitary cells, T3SO4 failed to displace [125I]T3 from nuclear receptors in intact cells or soluble preparations. Thus, T3SO4 was not directly thyromimetic in either an isolated human membrane system or a pituitary cell system in which nuclear receptor occupancy correlates with GH synthesis. Thyroid hormones inhibit [3H]glycosaminoglycan synthesis by cultured human dermal fibroblasts, and T3SO4 displayed about 0.5% the activity of T3 at 72 h. Human fibroblasts contained roughly the same level of microsomal p-nitrophenyl sulfatase activity as that previously observed in hepatic microsomes. Propylthiouracil (50 mumol/L) did not affect the action of T3SO4, suggesting that deiodination was not important for this activity of T3SO4. Thus, it appears T3SO4 has no intrinsic biological activity, but, under certain circumstances, may be reactivated by desulfation.