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.

RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Gα(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling” (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

The regulators of G protein signaling (RGS) domains of RGS4, RGS10, and GAIP retain GTPase activating protein activity in vitro

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gi but not by Gs class α-subunits. All RGS proteins share a conserved 120-amino acid sequence termed the RGS domain. We have demonstrated that the RGS domains of RGS4, RGS10, and GAIP retain GTPase accelerating activity with the Gi class substrates Giα1, Goα, and Gzα in vitro. No regulatory activity of the RGS domains was detected for Gsα. Short deletions within the RGS domain of RGS4 destroyed GTPase activating protein activity and Giα1 substrate binding. Comparable protein–protein interactions between Giα1–GDP–AlF4− and the RGS domain or full-length RGS4 were detected using surface plasmon resonance.

Inhibition of regulator of G protein signaling function by two mutant RGS4 proteins

Regulators of G protein signaling (RGS) proteins limit the lifetime of activated (GTP-bound) heterotrimeric G protein α subunits by acting as GTPase-activating proteins (GAPs). Mutation of two residues in RGS4, which, based on the crystal structure of RGS4 complexed with Giα1-GDP-AlF4−, directly contact Giα1 (N88 and L159), essentially abolished RGS4 binding and GAP activity. Mutation of another contact residue (S164) partially inhibited both binding and GAP activity. Two other mutations, one of a contact residue (R167M/A) and the other an adjacent residue (F168A), also significantly reduced RGS4 binding to Giα1-GDP-AlF4−, but in addition redirected RGS4 binding toward the GTPγS-bound form. These two mutant proteins had severely impaired GAP activity, but in contrast to the others behaved as RGS antagonists in GAP and in vivo signaling assays. Overall, these results are consistent with the hypothesis that the predominant role of RGS proteins is to stabilize the transition state for GTP hydrolysis. In addition, mutant RGS proteins can be created with an altered binding preference for the Giα-GTP conformation, suggesting that efficient RGS antagonists can be developed.

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.

Plasma Membrane Localization of Gαz Requires Two Signals

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the α subunits are myristoylated and/or palmitoylated, whereas the γ chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled α subunit, αz. Wild-type αz (αz-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does αz-WT. Removal of the myristoylation site produced a mutant αz that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of βγ with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on αz-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on αz-WT. Together these results suggest that myristate and βγ promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on αz specific localization at the plasma membrane.

Selection of Gβ Subunits with Point Mutations That Fail to Activate Specific Signaling Pathways In Vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G Protein in Dictyostelium discoideum

In Dictyostelium discoideum, a unique Gβ subunit is required for a G protein–coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Gα2, and Gβ genes completely impair all these responses. To dissect specificity in Gβγ signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gβ genes and isolated Gβ alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gβ subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gβγ by making point mutations on Gβ. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gβs and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtβγ interacts with its regulators, Gα and phosducin.

Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis

We have used the ciliate Euplotes to study the role of DNA polymerase in telomeric C strand synthesis. Euplotes provides a unique opportunity to study C strand synthesis without the complication of simultaneous DNA replication because millions of new telomeres are made at a stage in the life cycle when no general DNA replication takes place. Previously we showed that the C-strands of newly synthesized telomeres have a precisely controlled length while the G-strands are more heterogeneous. This finding suggested that, although synthesis of the G-strand (by telomerase) is the first step in telomere addition, a major regulatory step occurs during subsequent C strand synthesis. We have now examined whether G- and C strand synthesis might be regulated coordinately rather than by two independent mechanisms. We accomplished this by determining what happens to G- and C strand length if C strand synthesis is partially inhibited by aphidicolin. Aphidicolin treatment caused a general lengthening of the G-strands and a large increase in C strand heterogeneity. This concomitant change in both the G- and C strand length indicates that synthesis of the two strands is coordinated. Since aphidicolin is a very specific inhibitor of DNA polα and polδ, our results suggest that this coordinate length regulation is mediated by DNA polymerase.

Organization of G Proteins and Adenylyl Cyclase at the Plasma Membrane

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein α and β subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein α subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel

Inwardly rectifying potassium (K+) channels gated by G proteins (Kir3.x family) are widely distributed in neuronal, atrial, and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials, slowing the heart rate and modulating hormone release. They are directly activated by Gβγ subunits released from G protein heterotrimers of the Gi/o family upon appropriate receptor stimulation. Here we examine the role of isoforms of pertussis toxin (PTx)-sensitive G protein α subunits (Giα1–3 and GoαA) in mediating coupling between various receptor systems (A1, α2A, D2S, M4, GABAB1a+2, and GABAB1b+2) and the cloned counterpart of the neuronal channel (Kir3.1+3.2A). The expression of mutant PTx-resistant Gi/oα subunits in PTx-treated HEK293 cells stably expressing Kir3.1+3.2A allows us to selectively investigate that coupling. We find that, for those receptors (A1, α2A) known to interact with all isoforms, Giα1–3 and GoαA can all support a significant degree of coupling to Kir3.1+3.2A. The M4 receptor appears to preferentially couple to Giα2 while another group of receptors (D2S, GABAB1a+2, GABAB1b+2) activates the channel predominantly through Gβγ liberated from GoA heterotrimers. Interestingly, we have also found a distinct difference in G protein coupling between the two splice variants of GABAB1. Our data reveal selective pathways of receptor activation through different Gi/oα isoforms for stimulation of the G protein-gated inwardly rectifying K+ channel.

GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of Gαi subunits

GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of G-protein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein Gαi3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [35S]methionine, two pools of GAIP—a soluble and a membrane-anchored pool—were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membrane-anchored GAIP might be palmitoylated. We found that after labeling with [3H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the Gαi subfamily, Gαi1, Gαi2, Gαi3, Gαz, and Gαo, but not with members of other Gα subfamilies, Gαs, Gαq, and Gα12/13. The C terminus of Gαi3 is important for binding because a 10-aa C-terminal truncation and a point mutant of Gαi3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of Gαi3, which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP’s possible involvement in membrane trafficking.

A regulator of G protein signaling interaction surface linked to effector specificity

Proteins of the regulator of G protein signaling (RGS) family accelerate GTP hydrolysis by the α subunits (Gα) of G proteins, leading to rapid recovery of signaling cascades. Many different RGS proteins can accelerate GTP hydrolysis by an individual Gα, and GTP hydrolysis rates of different Gαs can be enhanced by the same RGS protein. Consequently, the mechanisms for specificity in RGS regulation and the residues involved remain unclear. Using the evolutionary trace (ET) method, we have identified a cluster of residues in the RGS domain that includes the RGS-Gα binding interface and extends to include additional functionally important residues on the surface. One of these is within helix α3, two are in α5, and three are in the loop connecting α5 and α6. A cluster of surface residues on Gα previously identified by ET, and composed predominantly of residues from the switch III region and helix α3, is spatially contiguous with the ET-identified residues in the RGS domain. This cluster includes residues proposed to interact with the γ subunit of Gtα's effector, cGMP phosphodiesterase (PDEγ). The proximity of these clusters suggests that they form part of an interface between the effector and the RGS-Gα complex. Sequence variations in these residues correlate with PDEγ effects on GTPase acceleration. Because ET identifies residues important for all members of a protein family, these residues likely form a general site for regulation of G protein-coupled signaling cascades, possibly by means of effector interactions.

Fidelity of G protein β-subunit association by the G protein γ-subunit-like domains of RGS6, RGS7, and RGS11

Several regulators of G protein signaling (RGS) proteins contain a G protein γ-subunit-like (GGL) domain, which, as we have shown, binds to Gβ5 subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human RGS6. When RGS6 is coexpressed with different Gβ subunits, only RGS6 and Gβ5 interact. The expression of mRNA for RGS6 and Gβ5 in human tissues overlaps. Predictions of α-helical and coiled-coil character within GGL domains, coupled with measurements of Gβ binding by GGL domain mutants, support the contention that Gγ-like regions within RGS proteins interact with Gβ5 subunits in a fashion comparable to conventional Gβ/Gγ pairings. Mutation of the highly conserved Phe-61 residue of Gγ2 to tryptophan, the residue present in all GGL domains, increases the stability of the Gβ5/Gγ2 heterodimer, highlighting the importance of this residue to GGL/Gβ5 association.

A molecular mechanism for signaling between seven-transmembrane receptors: evidence for a redistribution of G proteins

Although activation of one seven-transmembrane receptor can influence the response of a separate seven-transmembrane receptor, e.g., the phenomenon of synergism, the underlying mechanism(s) for this signaling process is unclear. The present study investigated communication between two receptors that exhibit classical synergism, e.g., human platelet thrombin and thromboxane A2 receptors. Activation of thrombin receptors caused an increase in ligand affinity of thromboxane A2 receptors. This effect (i) was shown to be specific, since a similar increase in ligand affinity was not caused by ADP or A23187; (ii) did not require cytosolic components, e.g., kinases, proteases, phosphatases, etc., because it occurred in isolated platelet membranes; (iii) was G protein-mediated because it was blocked by an Gαq C terminus antibody; and (iv) was associated with a net increase in Gαq coupling to thromboxane A2 receptors. Collectively, these data provide evidence that seven-transmembrane receptors that share a common Gα subunit can communicate with each other via a redistribution of their G proteins. Thus, activation of thrombin receptors increases Gαq association with thromboxane A2 receptors thereby shifting them to a higher affinity state. This signaling phenomenon, which modulates receptor-ligand affinity, may serve as a molecular mechanism for cellular adaptive processes such as synergism.