Direct interaction of Gβγ with a C-terminal Gβγ-binding domain of the Ca2+ channel α1 subunit is responsible for channel inhibition by G protein-coupled receptors

Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its βγ dimer (Gβγ). We report below the existence of two Gβγ-binding sites on the A-, B-, and E-type α1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gβγ-binding regions also bind the Ca2+ channel β subunit (CCβ), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in α1E of loop 1 with that of the G protein-insensitive and Gβγ-binding-negative loop 1 of α1C did not abolish inhibition by G proteins, but the exchange of the α1E C terminus with that of α1C did. This and properties of α1E C-terminal truncations indicated that the Gβγ-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gβγ to this site was inhibited by an α1-binding domain of CCβ, thus providing an explanation for the functional antagonism existing between CCβ and G protein inhibition. The data do not support proposals that Gβγ inhibits α1 function by interacting with the site located in the loop I–II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

The small GTP-binding protein Rho links G protein-coupled receptors and Gα12 to the serum response element and to cellular transformation

Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Gαs and Gαi2 families were found in human tumors; and members of the Gαq and Gα12 families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins we have focused recently on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, we have observed that activation of this transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-fos protooncogene. One of the c-fos promoter elements, the serum response element (SRE), plays a central regulatory role, and activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. With the aid of reporter plasmids for gene expression, we observed here that stimulation of m1 muscarinic acetylcholine receptors potently induced SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Gα12 family of heterotrimeric G protein α subunits strongly induced the SRE, while Gβ1γ2 dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, Gα12 and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation.

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of Gαi-coupled receptors

Chemotaxis is mediated by activation of seven-transmembrane domain, G protein-coupled receptors, but the signal transduction pathways leading to chemotaxis are poorly understood. To identify G proteins that signal the directed migration of cells, we stably transfected a lymphocyte cell line (300-19) with G protein-coupled receptors that couple exclusively to Gαq (the m3 muscarinic receptor), Gαi (the κ-opioid receptor), and Gαs (the β-adrenergic receptor), as well as the human thrombin receptor (PAR-1) and the C-C chemokine receptor 2B. Cells expressing receptors that coupled to Gαi, but not to Gαq or Gαs, migrated in response to a concentration gradient of the appropriate agonist. Overexpression of Gα transducin, which binds to and inactivates free Gβγ dimers, completely blocked chemotaxis although having little or no effect on intracellular calcium mobilization or other measures of cell signaling. The identification of Gβγ dimers as a crucial intermediate in the chemotaxis signaling pathway provides further evidence that chemotaxis of mammalian cells has important similarities to polarized responses in yeast. We conclude that chemotaxis is dependent on activation of Gαi and the release of Gβγ dimers, and that Gαi-coupled receptors not traditionally associated with chemotaxis can mediate directed migration when they are expressed in hematopoietic cells.

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent “precoupling” of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.

Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the α2A adrenergic receptor (α2A AR), with ERK activation mediated by the β2 adrenergic receptor (β2 AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the α2A AR, like that mediated by both the β2 AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant β-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both α2A and β2 AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the β2 AR, the α2A AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although β2 AR stimulation leads to redistribution of both the β2 AR and EGFR, activation of the α2A AR leads to redistribution of the EGFR but the α2A AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.

G protein subunits and the stimulation of phospholipase C by Gs-and Gi-coupled receptors: Lack of receptor selectivity of Galpha(16) and evidence for a synergic interaction between Gbeta gamma and the alpha subunit of a receptor activated G protein.

Stimulatory guanine nucleotide binding protein (Gs)-coupled receptors activated by luteinizing hormone, vasopressin, and the catecholamine isoproterenol (luteinizing hormone receptor, type 2 vasopressin receptor, and types 1 and 2 beta-adrenergic receptors) and the Gi-coupled M2 muscarinic receptor (M2R) were expressed transiently in COS cells, alone and in combination with Gbeta gamma dimers, their corresponding Galphas (Galpha(s), or Galpha(i3)) and either Galpha(q) or Galpha(16). Phospholipase C (PLC) activity, assessed by inositol phosphate production from preincorporated myo[3H]inositol, was then determined to gain insight into differential coupling preferences among receptors and G proteins. The following were observed: (i) All receptors tested were able to stimulate PLC activity in response to agonist occupation. The effect of the M2R was pertussis toxin sensitive. (ii) While, as expected, expression of Galpha(q) facilitated an agonist-induced activation of PLC that varied widely from receptor to receptor (400% with type 2 vasopressin receptor and only 30% with M2R), expression of Galpha(16) facilitated about equally well the activation of PLC by any of the tested receptors and thus showed little if any discrimination for one receptor over another. (iii) Gbeta gamma elevated basal (agonist independent) PLC activity between 2- and 4-fold, confirming the proven ability of Gbeta gamma to stimulate PLCbeta. (iv) Activation of expressed receptors by their respective ligands in cells coexpressing excess Gbeta gamma elicited agonist stimulated PLC activities, which, in the case of the M2R, was not blocked by pertussis toxin (PTX), suggesting mediation by a PTX-insensitive PLC-stimulating Galpha subunit, presumably, but not necessarily, of the Gq family. (v) The effects of Gbeta gamma and the PTX-insensitive Galpha elicited by M2R were synergistic, suggesting the possibility that one or more forms of PLC are under conditional or dual regulation of G protein subunits such that stimulation by one sensitizes to the stimulation by the other.

G protein-coupled cholecystokinin-B/gastrin receptors are responsible for physiological cell growth of the stomach mucosa in vivo.

Many peptide hormone and neurotransmitter receptors belonging to the seven membrane-spanning G protein-coupled receptor family have been shown to transmit ligand-dependent mitogenic signals in vitro. However, the physiological roles of the mitogenic activity through G protein-coupled receptors in vivo remain to be elucidated. Here we have generated G protein-coupled cholecystokinin (CCK)-B/gastrin receptor deficient-mice by gene targeting. The homozygous mice showed a remarkable atrophy of the gastric mucosa macroscopically, even in the presence of severe hypergastrinemia. The atrophy was due to a decrease in parietal cells and chromogranin A-positive enterochromaffin-like cells expressing the H+,K(+)-ATPase and histidine decarboxylase genes, respectively. Oral administration of a proton pump inhibitor, omeprazole, which induced hypertrophy of the gastric mucosa with hypergastrinemia in wild-type littermates, did not eliminate the gastric atrophy of the homozygotes. These results clearly demonstrated that the G protein-coupled CCK-B/gastrin receptor is essential for the physiological as well as pathological proliferation of gastric mucosal cells in vivo.

Most central nervous system D2 dopamine receptors are coupled to their effectors by Go

We reported previously that Go-deficient mice develop severe neurological defects that include hyperalgesia, a generalized tremor, lack of coordination, and a turning syndrome somewhat reminiscent of unilateral lesions of the dopaminergic nigro-striatal pathway. By using frozen coronal sections of serially sectioned brains of normal and Go-deficient mice, we studied the ability of several G protein coupled receptors to promote binding of GTPγS to G proteins and the ability of GTP to promote a shift in the affinity of D2 dopamine receptor for its physiologic agonist dopamine. We found a generalized, but not abolished reduction in agonist-stimulated binding of GTPγS to frozen brain sections, with no significant left–right differences. Unexpectedly, the ability of GTP to regulate the binding affinity of dopamine to D2 receptors (as seen in in situ [35S]sulpiride displacement curves) that was robust in control mice, was absent in Go-deficient mice. The data suggest that most of the effects of the Gi/Go-coupled D2 receptors in the central nervous system are mediated by Go instead of Gi1, Gi2, or Gi3. In agreement with this, the effect of GTP on dopamine binding to D2 receptors in double Gi1 plus Gi2- and Gi1 plus Gi3-deficient mice was essentially unaffected.

Crystal structure of the ectodomain of Methuselah, a Drosophila G protein-coupled receptor associated with extended lifespan

The Drosophila mutant methuselah (mth) was identified from a screen for single gene mutations that extended average lifespan. Mth mutants have a 35% increase in average lifespan and increased resistance to several forms of stress, including heat, starvation, and oxidative damage. The protein affected by this mutation is related to G protein-coupled receptors of the secretin receptor family. Mth, like secretin receptor family members, has a large N-terminal ectodomain, which may constitute the ligand binding site. Here we report the 2.3-Å resolution crystal structure of the Mth extracellular region, revealing a folding topology in which three primarily β-structure-containing domains meet to form a shallow interdomain groove containing a solvent-exposed tryptophan that may represent a ligand binding site. The Mth structure is analyzed in relation to predicted Mth homologs and potential ligand binding features.

Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor.

The alpha-factor pheromone receptor stimulates MATa yeast cells to undergo conjugation. The receptor contains seven transmembrane domains that function in ligand binding and in transducing a signal to the cytoplasmic receptor sequences to mediate G protein activation. A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor. The Pro-258-->Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor. Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to > or = 90% of the maximum level. Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6. The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling. These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor. Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation. Greater than 90% of all G protein-coupled receptors contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors.

The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

Rapid endocytosis of a G protein-coupled receptor: substance P evoked internalization of its receptor in the rat striatum in vivo.

Studies on cultured cells have shown that agonists induce several types of G protein-coupled receptors to undergo internalization. We have investigated this phenomenon in rat striatum, using substance P (SP)-induced internalization of the SP receptor (SPR) as our model system. Within 1 min of a unilateral striatal injection of SP in the anesthetized rat, nearly 60% of the SPR-immunoreactive neurons within the injection zone display massive internalization of the SPR--i.e., 20-200 SPR+ endosomes per cell body. Within the dendrites the SPR undergoes a striking translocation from the plasma membrane to endosomes, and these dendrites also undergo a morphological reorganization, changing from a structure of rather uniform diameter to one characterized by large, swollen varicosities connected by thin fibers. In both cell bodies and dendrites the number of SPR+ endosomes returns to baseline within 60 min of SP injection. The number of neurons displaying substantial endosomal SPR internalization is dependent on the concentration of injected SP, and the SP-induced SPR internalization is inhibited by the nonpeptide neurokinin 1 receptor antagonist RP-67,580. These data demonstrate that in the central nervous system in vivo, SP induces a rapid and widespread SPR internalization in the cell bodies and dendrites and a structural reorganization of the dendrites. These results suggest that many of the observations that have been made on the internalization and recycling of G protein-coupled receptors in in vitro transfected cell systems are applicable to similar events that occur in the mammalian central nervous system in vivo.

Targeting Gβγ signaling in arterial vascular smooth muscle proliferation: A novel strategy to limit restenosis

Restenosis continues to be a major problem limiting the effectiveness of revascularization procedures. To date, the roles of heterotrimeric G proteins in the triggering of pathological vascular smooth muscle (VSM) cell proliferation have not been elucidated. βγ subunits of heterotrimeric G proteins (Gβγ) are known to activate mitogen-activated protein (MAP) kinases after stimulation of certain G protein-coupled receptors; however, their relevance in VSM mitogenesis in vitro or in vivo is not known. Using adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of Gβγ signaling (βARKct), we evaluated the role of Gβγ in MAP kinase activation and proliferation in response to several mitogens, including serum, in cultured rat VSM cells. Our results include the striking finding that serum-induced proliferation of VSM cells in vitro is mediated largely via Gβγ. Furthermore, we studied the effects of in vivo adenoviral-mediated βARKct gene transfer on VSM intimal hyperplasia in a rat carotid artery restenosis model. Our in vivo results demonstrated that the presence of the βARKct in injured rat carotid arteries significantly reduced VSM intimal hyperplasia by 70%. Thus, Gβγ plays a critical role in physiological VSM proliferation, and targeted Gβγ inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.