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Mitochondrial dynamics and trafficking are essential to provide the energy required for neurotransmission and neural activity. We investigated how G protein-coupled receptors (GPCRs) and G proteins control mitochondrial dynamics and trafficking. The activation of Gα q inhibited mitochondrial trafficking in neurons through a mechanism that was independent of the canonical downstream PLCβ pathway. Mitoproteome analysis revealed that Gα q interacted with the Eutherian-specific mitochondrial protein armadillo repeat-containing X-linked protein 3 (Alex3) and the Miro1/Trak2 complex, which acts as an adaptor for motor proteins involved in mitochondrial trafficking along dendrites and axons. By generating a CNS-specific Alex3 knockout mouse line, we demonstrated that Alex3 was required for the effects of Gα q on mitochondrial trafficking and dendritic growth in neurons. Alex3-deficient mice had altered amounts of ER stress response proteins, increased neuronal death, motor neuron loss, and severe motor deficits. These data revealed a mammalian-specific Alex3/Gα q mitochondrial complex, which enables control of mitochondrial trafficking and neuronal death by GPCRs.

Kazrin is a protein widely expressed in vertebrates whose depletion causes a myriad of developmental defects, in part derived from altered cell adhesion and migration, as well as failure to undergo epidermal to mesenchymal transition. However, the primary molecular role of kazrin, which might contribute to all these functions, has not been elucidated yet. We previously identified one of its isoforms, kazrin C, as a protein that potently inhibits clathrin-mediated endocytosis when overexpressed. We now generated kazrin knock-out mouse embryonic fibroblasts to investigate its endocytic function. We found that kazrin depletion delays juxtanuclear enrichment of internalized material, indicating a role in endocytic traffic from early to recycling endosomes. Consistently, we found that the C-terminal domain of kazrin C, predicted to be an intrinsically disordered region, directly interacts with several early endosome (EE) components, and that kazrin depletion impairs retrograde motility of these organelles. Further, we noticed that the N-terminus of kazrin C shares homology with dynein/dynactin adaptors and that it directly interacts with the dynactin complex and the dynein light intermediate chain 1. Altogether, the data indicate that one of the primary kazrin functions is to facilitate endocytic recycling by promoting dynein/dynactin-dependent transport of EEs or EE-derived transport intermediates to the recycling endosomes., Competing Interests: IH, JR, AB, HG, MF, ER, AA No competing interests declared, MG Reviewing editor, eLife, (© 2023, Hernandez-Perez et al.)

All processes in human physiology relies on homeostatic mechanisms which require the activation of specific control circuits to adapt the changes imposed by external stimuli. One of the critical modulators of homeostatic balance is autophagy, a catabolic process that is responsible of the destruction of long-lived proteins and organelles through a lysosome degradative pathway. Identification of the mechanism underlying autophagic flux is considered of great importance as both protective and detrimental functions are linked with deregulated autophagy. At the mechanistic and regulatory levels, autophagy is activated in response to diverse stress conditions (food deprivation, hyperthermia and hypoxia), even a novel perspective highlight the potential role of physical forces in autophagy modulation. To understand the crosstalk between all these controlling mechanisms could give us new clues about the specific contribution of autophagy in a wide range of diseases including vascular disorders, inflammation and cancer. Of note, any homeostatic control critically depends in at least two additional and poorly studied interdependent components: a receptor and its downstream effectors. Addressing the selective receptors involved in autophagy regulation is an open question and represents a new area of research in this field. G-protein coupled receptors (GPCRs) represent one of the largest and druggable targets membrane receptor protein superfamily. By exerting their action through G proteins, GPCRs play fundamental roles in the control of cellular homeostasis. Novel studies have shown Gαq, a subunit of heterotrimeric G proteins, as a core modulator of mTORC1 and autophagy, suggesting a fundamental contribution of Gαq-coupled GPCRs mechanisms in the control of this homeostatic feedback loop. To address how GPCR-G proteins machinery integrates the response to different stresses including oxidative conditions and mechanical stimuli, could provide deeper insight into new signaling pathways and open potential and novel therapeutic strategies in the modulation of different pathological conditions.

The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular homeostasis upon nutrient fluctuations., (© 2021. The Author(s).)

Background: Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by uncontrolled proliferation of precursor myeloid-lineage cells in the bone marrow. AML is also characterized by patients with poor long-term survival outcomes due to relapse. Many efforts have been made to understand the biological heterogeneity of AML and the challenges to develop new therapies are therefore enormous. G Protein-coupled Receptors (GPCRs) are a large attractive drug-targeted family of transmembrane proteins, and aberrant GPCR expression and GPCR-mediated signaling have been implicated in leukemogenesis of AML. This review aims to identify the molecular players of GPCR signaling, focusing on the hematopoietic system, which are involved in AML to help developing novel drug targets and therapeutic strategies., Methods: We undertook an exhaustive and structured search of bibliographic databases for research focusing on GPCR, GPCR signaling and expression in AML., Results and Conclusion: Many scientific reports were found with compelling evidence for the involvement of aberrant GPCR expression and perturbed GPCR-mediated signaling in the development of AML. The comprehensive analysis of GPCR in AML provides potential clinical biomarkers for prognostication, disease monitoring and therapeutic guidance. It will also help to provide marker panels for monitoring in AML. We conclude that GPCR-mediated signaling is contributing to leukemogenesis of AML, and postulate that mass spectrometrybased protein profiling of primary AML cells will accelerate the discovery of potential GPCR related biomarkers for AML., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Proper endosomal trafficking of ligand-activated G-protein-coupled receptors (GPCRs) is essential to spatiotemporally tune their physiological responses. For the monocyte chemoattractant receptor 2 (CCR2B; one of two isoforms encoded by CCR2), endocytic recycling is important to sustain monocyte migration, whereas filamin A (FLNa) is essential for CCL2-induced monocyte migration. Here, we analyze the role of FLNa in the trafficking of CCR2B along the endocytic pathway. In FLNa-knockdown cells, activated CCR2B accumulated in enlarged EEA-1-positive endosomes, which exhibited slow movement and fast fluorescence recovery, suggesting an imbalance between receptor entry and exit rates. Utilizing super-resolution microscopy, we observed that FLNa-GFP, CCR2B and β2-adrenergic receptor (β2AR) were present in actin-enriched endosomal microdomains. Depletion of FLNa decreased CCR2B association with these microdomains and concomitantly delayed CCR2B endosomal traffic, without apparently affecting the number of microdomains. Interestingly, CCR2B and β2AR signaling induced phosphorylation of FLNa at residue S2152, and this phosphorylation event was contributes to sustain receptor recycling. Thus, our data strongly suggest that CCR2B and β2AR signals to FLNa to stimulate its endocytosis and recycling to the plasma membrane., (© 2017. Published by The Company of Biologists Ltd.)

The guanine nucleotide exchange factor Rgnef (also known as ArhGEF28 or p190RhoGEF) promotes colon carcinoma cell motility and tumor progression via interaction with focal adhesion kinase (FAK). Mechanisms of Rgnef activation downstream of integrin or G protein-coupled receptors remain undefined. In the absence of a recognized G protein signaling homology domain in Rgnef, no proximal linkage to G proteins was known. Utilizing multiple methods, we have identified Rgnef as a new effector for Gα13 downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of Gα13, gastrin-induced FAK Tyr(P)-397 and paxillin Tyr(P)-31 phosphorylation were reduced. RhoA GTP binding and promoter activity were increased by Rgnef in combination with active Gα13. Rgnef co-immunoprecipitated with activated Gα13Q226L but not Gα12Q229L. The Rgnef C-terminal (CT, 1279-1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented Gα13-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to Gα13. Point mutations of Rgnef-CT residues disrupt association with active Gα13 but not Gαq. These results show that Rgnef functions as an effector of Gα13 signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In the last few years the interactome of Gαq has expanded considerably, contributing to improve our understanding of the cellular and physiological events controlled by this G alpha subunit. The availability of high-resolution crystal structures has led the identification of an effector-binding region within the surface of Gαq that is able to recognise a variety of effector proteins. Consequently, it has been possible to ascribe different Gαq functions to specific cellular players and to identify important processes that are triggered independently of the canonical activation of phospholipase Cβ (PLCβ), the first identified Gαq effector. Novel effectors include p63RhoGEF, that provides a link between G protein-coupled receptors and RhoA activation, phosphatidylinositol 3-kinase (PI3K), implicated in the regulation of the Akt pathway, or the cold-activated TRPM8 channel, which is directly inhibited upon Gαq binding. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has also been described as a novel PLCβ-independent signalling axis that relies upon the interaction between this G protein and two novel effectors (PKCζ and MEK5). Additionally, the association of Gαq with different regulatory proteins can modulate its effector coupling ability and, therefore, its signalling potential. Regulators include accessory proteins that facilitate effector activation or, alternatively, inhibitory proteins that downregulate effector binding or promote signal termination. Moreover, Gαq is known to interact with several components of the cytoskeleton as well as with important organisers of membrane microdomains, which suggests that efficient signalling complexes might be confined to specific subcellular environments. Overall, the complex interaction network of Gαq underlies an ever-expanding functional diversity that puts forward this G alpha subunit as a major player in the control of physiological functions and in the development of different pathological situations., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Contrary to previous assumptions, G proteins do not permanently reside on the plasma membrane, but are constantly monitoring the cytoplasmic surfaces of the plasma membrane and endomembranes. Here, we report that the Gαq and Gα11 proteins locate at the mitochondria and play a role in a complex signaling pathway that regulates mitochondrial dynamics. Our results provide evidence for the presence of the heteromeric G protein (Gαq/11βγ) at the outer mitochondrial membrane and for Gαq at the inner membrane. Both localizations are necessary to maintain the proper equilibrium between fusion and fission; which is achieved by altering the activity of mitofusin proteins, Drp1, OPA1 and the membrane potential at both the outer and inner mitochondrial membranes. As a result of the absence of Gαq/11, there is a decrease in mitochondrial fusion rates and a decrease in overall respiratory capacity, ATP production and OXPHOS-dependent growth. These findings demonstrate that the presence of Gαq proteins at the mitochondria serves as a physiological function: stabilizing elongated mitochondria and regulating energy production in Drp1 and Opa1 dependent mechanisms. This thereby links organelle dynamics and physiology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Filamin A (FLNa) is an actin-crosslinking protein necessary for stabilizing the cell surface, organizing protrusive activity and for promoting efficient cellular translocation. Recently, our group demonstrated the requirement of FLNa for the internalization of the chemokine receptor CCR2B., Methodology and Principal Findings: In order to study the role of FLNa in vitro and in real-time, we have developed a fluorescent FLNa-EGFP construct. In this novel imaging tool, we introduced the EGFP-tag inside the flexible hinge 1 region of FLNa between two calpain cleavage sites. Our findings indicate that the FLNa-EGFP construct was correctly expressed, cleaved by calpain and colocalized with actin filaments as shown by immunostaining experiments in the human melanoma cell lines A7 (FLNa-repleted) and M2 (FLNa-deficient). In addition, scanning-electron microscopy (SEM) and micropatterning studies also provided clear evidence that the cell rigidity was restored. FLNa-EGFP allowed us to demonstrate the interaction of FLNa with the chemokine receptor CCR2B in endocytic vesicles after CCL2 ligand stimulation. Through live-cell imaging studies we show that the CCR2B receptor in Rab5-positive vesicles moves along filamin A-positive fibers., Significance: Taken together, these results outline the functionality of the FLNa-EGFP and the importance of filamin A for receptor internalization and movement into endocytic vesicles.

p120 Catenin (p120(ctn)) regulates cadherin stability, and thus facilitates strong cell-cell adhesion. Previously, we demonstrated that Gα(12) interacts with p120(ctn). In the present study, we have delineated a region of p120(ctn) that binds to Gα(12). We report that the N-terminal region of p120(ctn) (amino acids 1-346) is necessary and sufficient for the interaction. While the coiled-coiled domain and a charged region, comprising a.a 102-120, were found to be dispensable, amino acids 121-323 were required for p120(ctn) binding to Gα(12). This region harbors the phosphorylation domain of p120(ctn) and has been postulated as important for RhoA regulation. Downregulation of Src family kinase-induced tyrosine phosphorylation of p120(ctn) was observed in the presence of activated Gα(12). This down-regulation was triggered by three different Gα(12) mutants uncoupled from RhoA signalling. Furthermore, a dominant active form of RhoA did not reduce Src-induced phosphoryaltion of p120(ctn). In summary, our results suggest that Gα(12) binds to p120(ctn) and modulates its phosphorylation status through a Rho-independent mechanism. Gα(12) emerges as an important regulator of p120(ctn) function, and possibly of cadherin-mediated adhesion and/or cell motility., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells produced a significant reduction of the extracellular signal-regulated kinase (ERK) response to CCL2. This effect is independent of its role in receptor phosphorylation because the kinase-deficient mutant GRK2K220R was able to reduce this response, and ERK activation by CCR2BIX, a phosphorylation-defective receptor mutant, was also inhibited by GRK2. Constructs containing the Galpha(q)-binding RGS-like RH domain of GRK2 or its Gbetagamma-binding domain could not reproduce the inhibition, thus revealing that GRK2 acts downstream of G proteins. Interestingly, chemokine-driven mitogen-activated protein kinase kinase (MEK) stimulation is not affected in cells overexpressing GRK2 or GRK2K220R or in splenocytes from heterozygous GRK2 mice, where reduced kinase levels correlate with enhanced ERK activation by chemokines. We find GRK2 and MEK in the same multimolecular complex, thus suggesting a mechanism for GRK2 regulation of ERK activity that involves a direct or coordinate interaction with MEK. These results suggest an important role for GRK2 in the control of chemokine induction of ERK activation at the level of the MEK-ERK interface.

Metabolically unstable proteins are involved in a multitude of regulatory networks, including those that control cell signaling, the cell cycle and in many responses to physiological stress. In the present study, we have determined the stability and characterized the degradation process of some members of the G(q) class of heterotrimeric G proteins. Pulse-chase experiments in HEK293 cells indicated a rapid turnover of endogenously expressed Galpha(q) and overexpressed Galpha(q) and Galpha(16) subunits. Pretreatment with proteasome inhibitors attenuated the degradation of both G alpha subunits. In contrast, pretreatment of cells with inhibitors of lysosomal proteases and nonproteasomal cysteine proteases had very little effect on the stability of the proteins. Significantly, the turnover of these proteins is not affected by transient activation of their associated receptors. Fractionation studies showed that the rates of Galpha(q) and Galpha16 degradation are accelerated in the cytosol. In fact, we show that a mutant Galpha(q) which lacks its palmitoyl modification site, and which is localized almost entirely in the cytoplasm, has a marked increase in the rate of degradation. Taken together, these results suggest that the G(q) class proteins are degraded through the proteasome pathway and that cellular localization and/or other protein interactions determine their stability.

The catenin p120 (p120ctn) is an armadillo repeat domain protein that binds to cadherins and has been shown to facilitate strong cell-cell adhesion. We have investigated a possible link between heterotrimeric G proteins and p120ctn, and found that both Galpha12 and Galpha13 can completely and selectively abrogate the p120ctn-induced branching phenotype in different cell types. Consistent with these observations, the expression of Galpha12 or Galpha13 compensates for the reduction of Rho activity induced by p120ctn. On the other hand, p120ctn can be selectively coimmunoprecipitated with Galpha12, and the coimmunoprecipitation was favored by activation of the G protein. A specific interaction between p120ctn and Galpha12Q231L was also observed in in vitro binding experiments. In addition, p120ctn can be immunoprecipitated along with Galpha12Q231L in L cells in absence of E-cadherin. Interestingly, the expression of Galpha12Q231L increases the amount of p120ctn associated with E-cadherin. These findings demonstrate that Galpha12 and p120ctn are binding partners, and they also suggest a role for Galpha12 in regulating p120ctn activity and its interaction with cadherins. We propose that the Galpha12-p120ctn interaction acts as a molecular switch, which regulates cadherin-mediated cell-cell adhesion.

G protein-coupled receptors (GPCRs) initiate diverse down-stream signaling events in response to ligand stimulation, as rapid activation of the extracellular signal-regulated kinase ERK1 and ERK2. The chemokine monocyte chemoattractant protein-1 (MCP-1) is the agonist for several chemokine receptors that belong to the GPCR superfamily, CCR2 being the most important. Stimulation of mitogen-activated protein kinases (MAPKs) by MCP-1 has been implicated in integrin activation and chemotaxis, but the molecular pathways down-stream of the receptors remain unclear. To dissect the cascade of events leading to MAPK activation upon CCR2 receptor stimulation, several specific inhibitors and mutants of signal transduction proteins were used in monocytic cells endogenously expressing CCR2 and/or in human embryonic kidney-293 cells transfected with CCR2B receptors and epitope-tagged ERK1. We show that ERK activation by MCP-1 involves heterotrimeric Gi protein subunits, protein kinase C, phosphoinositide-3-kinase, and Ras. On the other hand, the activity of cytosolic tyrosine kinases, epidermal growth factor receptor transactivation, or variations in intracellular calcium levels are not required for the mitogenic activation elicited by MCP-1. In addition, we find that internalization of CCR2B itself is not necessary for efficient MCP-1-induced activation of ERK, although a dynamin mutant partially inhibits ERK stimulation. These results suggest that different parallel pathways are being activated that lead to the full activation of the mitogen-activated protein kinase cascade and that internalization of other signaling proteins but not of the receptor is required for complete ERK activation.

Recent evidence demonstrates that the alpha subunits of some heterotrimeric GTP-binding proteins (G proteins) are subject to modification by protein kinase C (PKC). For the family of G proteins that activate the phospholipase C/inositol trisphosphate/calcium/PKC pathway, such modification could result in G protein autoregulation. To examine the potential regulation of members of the Galphaq family by PKC phosphorylation, we expressed the thyrotropin-releasing hormone (TRH) receptor in combination with Galphaq, Galpha11, Galpha14, Galpha15, or Galpha16 in Xenopus oocytes and examined the regulation of signaling by PKC activators and inhibitors. For Galpha16 and Galpha15, the two family members of hematopoietic lineage, PKC activators reduce both the magnitude and the time course of TRH-mediated responses; PKC inhibitors have the opposite effect. The PKC-mediated effects are evident in measurements of GTPase activity, suggesting that the regulation is occurring early in the signaling pathway. In vivo phosphorylation experiments demonstrate that Galpha16 is a substrate for PKC modification. By comparison, Galphaq is not phosphorylated by PKC in vivo, and oocytes expressing Galphaq are not functionally modulated by PKC. Repeated TRH stimulation of oocytes expressing Galpha16 mimics the effects of PKC activators, and this functional regulation is correlated with an increase in Galpha16 phosphorylation. A mutant Galpha16 with four consensus PKC phosphorylation sites removed is not phosphorylated in vivo, and TRH responses mediated through the mutant are not regulated by PKC. These results demonstrate that signaling involving hematopoietic G proteins is subject to PKC-mediated autoregulation, at least in part, by phosphorylation of the G protein alpha subunit.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in determining the rate and extent of G protein-coupled receptor (GPCR) desensitization and resensitization. Recent data indicate that GRK2 activity, subcellular distribution and expression are tightly regulated. The important physiological function of GRK2 as a modulator of the efficacy of GPCR signal transduction systems is exemplified by its relevance in cardiovascular physiopathology as well as by its emerging role in the regulation of chemokine receptors.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called beta-adrenergic kinase 1 or betaARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein beta-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant betaARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines.

Expression of G protein alpha subunits of the Gq family with various G protein-coupled receptors induces activation of an inositol 1,4, 5-trisphosphate (IP3)/Ca2+-mediated Cl- conductance in Xenopus oocytes. Our present data show that two members of this family, the human Galpha16 subunit and the murine homologue Galpha15, can induce both activation and inhibition of these agonist-induced currents. Although extremely low amounts (10-50 pg) of injected Galpha16 subunit cRNA cause modest ( approximately 2-fold) enhancement of ligand-induced Cl- currents in oocytes co-injected with thyrotropin-releasing hormone (TRH) receptor cRNA 48 h postinjection, larger Galpha16 and Galpha15 cRNA injections cause >10-fold inhibition of TRH or 5HT2c receptor responses. The inhibition is analyzed in this study. The inhibited currents are recovered if various Gbetagamma subunit combinations are also expressed with the Galpha subunits. The constitutively active mutant, Galpha16Q212L, also causes a strong attenuation of the ligand-induced Cl- currents, but this inhibition is not recovered by co-expression of Gbetagamma subunits. These results indicate that the free Galpha subunit is responsible for the inhibitory signal. Although expression of TRH receptor alone produces maximum responses approximately 48 h after injection, co-expression of TRH receptor with Galpha16 results in enhanced responses 6-12 h postinjection, followed by complete attenuation at 36 h. Furthermore, injection of Galpha16 cRNA alone at comparable levels gives rise to spontaneous Cl- currents within 6-12 h postinjection, suggesting that the early spontaneous activation underlies the later suppression. Expression of other G protein alpha subunits of the Gq family, at cRNA levels considerably higher than effective for Galpha16, produces both analogous spontaneous Cl- currents and, later, inhibition of ligand-induced Cl- currents. Experiments with direct injection of IP3 and of Ca2+ suggest that this inhibition is consistent with the down-regulation of IP3 receptors. These data indicate that both enhancement and inhibition of signaling through G protein-coupled receptors can be mediated by the expression level and/or activity of an individual G protein.

The G protein beta subunit, Gbeta5, is predominantly expressed in the central nervous system. In rodent brain, Gbeta5 is expressed as a protein with an apparent molecular mass of 39,000 daltons (39 kDa). We have identified an additional Gbeta5 immunoreactive protein of apparent size 44 kDa in the vertebrate retina. Molecular cloning and sequencing of polymerase chain reaction products revealed that the cDNA encoding the larger species of Gbeta5 (Gbeta5L) was identical to the shorter form with the addition of 126 base pairs of 5' DNA sequence potentially encoding an in-frame 42-amino acid extension. Sequencing of mouse Gbeta5 genomic clones demonstrated that the 126-base pair of retinal-specific coding material is derived from a hitherto undetected 5' exon. During sucrose density gradient fractionation of bovine retinas, the 44-kDa Gbeta5L protein co-purified with rod outer segment membranes. Incubation of rod outer segment membranes with the nonhydrolyzable guanine nucleotide, GTPgammaS (guanosine 5'-3-O-(thio)triphosphate), which released the Gbeta subunit of transducin (Gbeta1), failed to remove Gbeta5L. The 39-kDa Gbeta5 protein displayed differential association with retinal and brain membranes. In the retina, Gbeta5 was present as a soluble protein and was undetectable in the membrane fraction, whereas in the brain approximately 70% of Gbeta5 was associated with cellular membranes. In transient COS-7 cell expression experiments, Gbeta5L formed functional Gbetagamma dimers and Galphabetagamma heterotrimers, and activated phosphoinositide-specific phospholipase Cbeta2 in a manner indistinguishable from the 39-kDa Gbeta5 protein. The cloning of the retinal-specific Gbeta5L cDNA suggests the existence of potentially novel G protein-mediated signaling cascades in photoreception.

In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G protein-coupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2+, diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxin-insensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that G alpha 12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells.

PTH-related protein activates a G protein-coupled PTH/PTHrP receptor in many cell types and produces diverse biological actions. To study the signal transduction events associated with biological activity of the PTH/PTHrP receptor in vascular smooth muscle, a principal PTHrP-responsive tissue, rat aortic smooth muscle cells (A10) were stably transfected with a plasmid encoding a PTH/PTHrP receptor and tested for ligand binding, PTHrP-(1-34)-induced cAMP levels, inositol phosphate production, and cytosolic calcium transients. Of nineteen G418-resistant lines recovered, all exhibited high affinity binding [approximately dissociation constant (Kd) > 10(-10)) of iodinated [Tyr36]hPTHrP(1-36)NH2 and ligand-induced cAMP accumulation (2- to 100-fold), which was directly proportional to PTH/PTHrP receptor number (range 4 x 10(3) to 7 x 10(7) sites/cell]. PTHrP had no effect on intracellular calcium or inositol phosphate formation in any cell line regardless of receptor number despite the presence of detectable G alpha q). Transient overexpression of individual G alpha q proteins (G alpha q, G alpha 11 or G alpha 14) into PTH/PTHrP receptor-expressing A10 cells conferred the ability of PTHrP to increase intracellular calcium and inositol phosphate formation. Ligand activation of the recombinant PTH/PTHrP receptor elicited appropriate downstream biological effects in A10 cells including inhibition of DNA synthesis and osteopontin messenger RNA (mRNA) expression. Thus, a single PTH/PTHrP receptor, though capable of coupling to different G proteins, signals exclusively through a cAMP-dependent pathway in vascular smooth muscle.

G-proteins of the alphaq family link extracellular stimulation of plasma membrane receptors to phospholipase C and consequently to intracellular Ca2+ release. Because they might function in initiating Ca2+ release at fertilization, we examined Galphaq family proteins in oocytes and eggs of Xenopus laevis. Three members of this protein family were identified by immunoblotting and antisense depletion. These proteins are barely detectable in the immature oocyte, but undergo a 6-fold increase in amount during oocyte maturation. This increase in Galphaq family protein expression correlates with the acquisition, during oocyte maturation, of the ability to release Ca2+ at fertilization (Schlichter and Elinson, 1981, Dev. Biol. 83, 33-41). In contrast, amounts of Galphas and Galphai3 are constant during maturation. We also examined the amounts of Galphaq, Galphas, and Galphai3 proteins during early development. While amounts of Galphas and Galphai3 show little or no change, Galphaq family protein expression increases 27-fold between the egg and neurula stages, suggesting that these proteins may be important in initiating Ca2+ release during early development.

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12 has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of G alpha 12 increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of G alpha 12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-G alpha 12 resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of G alpha o did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-G alpha 12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for G alpha 12 in the mitogenic response to thrombin in human astroglial cells.

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist-induced, Ca(2+)-activated Cl- currents that were measured using a two-electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX-sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor-activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase.

Nucleotide binding properties of the G protein alpha subunit G(o)alpha were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [35S]GTP gamma S (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G(o)alpha cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of G(o)alpha for GTP gamma S or Mg2+ were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21ras proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTP gamma S binding but retained the capacity to interact with the beta gamma subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type G(o)alpha increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl- response, suggesting that this mutation resulted in a dominant negative phenotype.

Thyrotropin-releasing hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cell membranes. The stimulation of the phosphoinositide phospholipase C (PI/PLC) activity can be blocked by incubation of GH3 membranes with polyclonal antibodies directed against a peptide derived from the C-terminal region of G alpha q and G alpha 11. Antibodies directed against the C-terminal region of other G alpha-subunits had no detectable effect. The inhibition was specific since addition of the peptide that was used to prepare the antibody completely reversed the inhibition. Further evidence for the coupling of the TRH receptor to G alpha q or G alpha 11 comes from a reconstitution experiment in which human embryonic kidney cells were transiently transfected with cDNAs corresponding to the TRH receptor, G alpha q or G alpha 11. The PIP2 hydrolysis detected with membranes from cells that over-expressed the TRH receptor alone was low, however, co-expression with the G alpha q or G alpha 11 subunits produced a synergistic stimulation of PI-PLC activity. In contrast, co-expression of these alpha-subunits with the M2 muscarinic acetylcholine receptor induced a weak stimulation of PIP2 hydrolysis. The results presented here suggest that the TRH-dependent stimulation of PI-PLC in GH3 cells is mediated through the G-protein alpha-subunits, G alpha q and/or G alpha 11.

The interaction of different histone oligomers with nucleosomes has been investigated by using nondenaturing gel electrophoresis. In the presence of 0.2 M NaCl, the addition of the pairs H2A,H2B or H3,H4 or the four core histones to nucleosome core particles produces a decrease in the intensity of the core particle band and the appearance of aggregated material at the top of the gel, indicating that all these histone oligomers are able to associate with nucleosomes. Equivalent results were obtained by using oligonucleosome core particles. Additional electrophoretic results, together with second-dimension analysis of histone composition and fluorescence and solubility studies, indicate that H2A,H2B, H3,H4, and the four core histones can migrate spontaneously from the aggregated nucleosomes containing excess histones to free core DNA. In all cases the estimated yield of histone transfer is very high. Furthermore, the results obtained from electron microscopy, solubility, and supercoiling assays demonstrate the transfer of excess histones from oligonucleosomes to free circular DNA. However, the extent of solubilization obtained in this case is lower than that observed with core DNA as histone acceptor. Our results demonstrate that nucleosome core particles can be formed in 0.2 M NaCl by the following mechanisms: (1) transfer of excess core histones from oligonucleosomes of free DNA, (2) transfer to excess H2A,H2B and H3,H4 associated separately with oligonucleosomes to free DNA, (3) transfer to excess H2A,H2B initially associated with oligonucleosomes to DNA, followed by the reaction of the resulting DNA-(H2A,H2B) complex with oligonucleosomes containing excess H3,H4, and (4) a two-step transfer reaction similar to that indicated in (3), in which excess histones H3,H4 are transferred to DNA before the reaction with oligonucleosomes containing excess H2A,H2B. The possible biological implications of these spontaneous reactions are discussed in the context of the present knowledge of the nucleosome function.

The increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) produced by core histones is higher than that produced by very lysine-rich histones (H1 and H5). In the presence of the anionic detergent sodium dodecyl sulfate (SDS) the enhancement of ANS fluorescence caused by these two groups of histones is roughly the same, but much lower than that observed for core histones in the absence of this detergent. However, the increase of ANS fluorescence produced by histone-SDS complexes is high enough to use it for the staining of these proteins separated in SDS-polyacrylamide gels. Histone bands are stained with ANS after electrophoresis and visualized by transillumination of the gel with a uv light source. The method described in this work allows the rapid detection of less than 0.5 microgram of histone per band.

In non-denaturing low ionic strength gels, the titration of core DNA with H2A,H2B produces five well-defined bands. Quantitative densitometry and cross-linking experiments indicate that these bands are due to the successive binding of H2A,H2B dimers to core DNA. Only two bands are obtained with DNA-(H3,H4) samples. The slower of these bands is broad and presumably corresponds to two complexes containing one and two H3,H4 tetramers, respectively. In gels of higher ionic strength, DNA-(H2A,H2B) samples produce an ill-defined band, suggesting that the lifetime of the complexes containing H2A,H2B is relatively short. However, the low intensity of the free DNA band observed in these gels indicates that most of the DNA is associated with H2A,H2B. In agreement with this, our results obtained using different techniques (sedimentation, cross-linking, trypsin and nuclease digestions, and thermal denaturation) demonstrate that the association of H2A,H2B with core DNA occurs in free solution in both the absence and presence of NaCl (0.1 to 0.2 M). The low mobilities of DNA-(H2A,H2B) complexes, together with sedimentation and DNase I digestion results, indicate that the DNA in these complexes is not folded into the compact structure found in the core particle. Furthermore, non-denaturing gels have been used to study the dynamic properties of DNA-(H2A,H2B) and DNA-(H3,H4) complexes in 0.2 M-NaCl. Our results show that: (1) H2A,H2B and H3,H4 can associate, respectively, with DNA-(H3,H4) and DNA-(H2A,H2B) to produce complexes containing the four core histones; (2) DNA-(H2A,H2B) and DNA-(H3,H4) are able to transfer histones to free core DNA; (3) an exchange of histone pairs takes place between DNA-(H2A,H2B) and DNA-(H3,H4) and produces complexes with the same histone composition as that of the normal nucleosome core particle; and (4) although both histone pairs can exchange, histones H2A,H2B show a higher tendency than H3,H4 to migrate from one incomplete core particle to another. The complexes produced in these reactions have the same compact structure as reconstituted core particles containing the four core histones. Our kinetic results are consistent with a reaction mechanism in which the transfer of histones involves direct contacts between the reacting complexes. The possible participation of these spontaneous reactions on the mechanism of nucleosome assembly is discussed.

In the present report it is shown that histone bands in urea-acetic acid or Triton-urea-acetic acid-polyacrylamide gels can be stained with the fluorescent dye 1-anilinonaphthalene 8-sulfonate and visualized by transillumination of the gel with an uv-light source. The 1-anilinonaphthalene 8-sulfonate staining method described here for urea and Triton-urea gels is rapid (it can be completed in 90 min) and allows the detection of less than 1 micrograms of histone per band.