Related Galpha subunits play opposing roles during Dictyostelium development

Of the several known Dictyostelium G protein subunits, the Galpha4 and Galpha5 subunits are the most closely related pair based on phylogenetic analysis and expression patterns, but these subunits perform different roles during development. To investigate potential relationships between these subunits with respect to cell differentiation, chimeric organisms composed of strains lacking or overexpressing either subunit were created and examined for developmental morphogenesis and spore production. Chimeras of galpha4 null and galpha5 null strains or Galpha4 and Galpha5 overexpression strains displayed compensatory morphogenesis, implying that the subunits promote complementary developmental processes. However, chimeras composed of galpha4 null and Galpha5 overexpression strains or galpha5 null and Galpha4 overexpression strains displayed distorted tip morphogenesis, suggesting the strains of these chimeras share common developmental deficiencies. Cells lacking the Galpha5 subunit localized to the prespore region of chimeras similar to the pattern observed for cells overexpressing the Galpha4 subunit, and cells overexpressing the Galpha5 subunit displayed localization patterns similar to galpha4 null mutants. A strain overexpressing both subunits displayed a partial suppression of morphology, gene expression, and cell localization phenotypes associated with the overexpression of the individual Galpha subunit genes, suggesting that each Galpha subunits can inhibit signaling mediated by the other subunit. Overexpression of the Galpha5 subunit inhibited chemotaxis and cGMP accumulation in response to folic acid, indicating that the Galpha5 subunit can inhibit early steps in the Galpha4-mediated signal transduction pathway. The contrasting phenotypes of the Galpha mutants suggest the Galpha4 and Galpha5 subunits provide opposing functions in cell differentiation, localization, and chemotactic responses to folic acid.     

Folic acid stimulation of the Gα4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium

In Dictyostelium discoideum, several G proteins are known to mediate the transduction of signals that direct chemotactic movement and regulate developmental morphogenesis. The G protein alpha subunit encoded by the Galpha4 gene has been previously shown to be required for chemotactic responses to folic acid, proper developmental morphogenesis, and spore production. In this study, cells overexpressing the wild type Galpha4 gene, due to high copy gene dosage (Galpha4HC), were found to be defective in the ability to form the anterior prestalk cell region, express prespore- and prestalk-cell specific genes, and undergo spore formation. In chimeric organisms, Galpha4HC prespore cell-specific gene expression and spore production were rescued by the presence of wild-type cells, indicating that prespore cell development in Galpha4HC cells is limited by the absence of an intercellular signal. Transplanted wild-type tips were sufficient to rescue Galpha4HC prespore cell development, suggesting that the rescuing signal originates from the anterior prestalk cells. However, the deficiencies in prestalk-specific gene expression were not rescued in the chimeric organisms. Furthermore, Galpha4HC cells were localized to the prespore region of these chimeric organisms and completely excluded from the anterior prestalk region, suggesting that the Galpha4 subunit functions cell-autonomously to prevent anterior prestalk cell development. The presence of exogenous folic acid during vegetative growth and development delayed anterior prestalk cell development in wild-type but not galpha4 null mutant aggregates, indicating that folic acid can inhibit cell-type-specific differentiation by stimulation of the Galpha4-mediated signal transduction pathway. The results of this study suggest that Galpha4-mediated signals can regulate cell-type-specific differentiation by promoting prespore cell development and inhibiting anterior prestalk-cell development.     

Galpha(olf) is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum

In the brain, dopamine and adenosine stimulate cyclic AMP (cAMP) production through D1 and A2a receptors, respectively. Using mutant mice deficient in the olfactory isoform of the stimulatory GTP-binding protein alpha subunit, Galpha(olf), we demonstrate here the obligatory role of this protein in the adenylyl cyclase responses to dopamine and adenosine in the caudate putamen. Responses to dopamine were also dramatically decreased in the nucleus accumbens but remained unaffected in the prefrontal cortex. Moreover, in the caudate putamen of mice heterozygous for the mutation, the amounts of Galpha(olf) were half of the normal levels, and the efficacy of dopamine- and CGS 21680 A(2) agonist-stimulated cAMP production was decreased. Together, these results identify Galpha(olf) as a critical parameter in the responses to dopamine and adenosine in the basal ganglia.     

Rgs1 regulates multiple Galpha subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism

Regulators of G-protein signaling (RGS proteins) negatively regulate heterotrimeric G-protein cascades that enable eukaryotic cells to perceive and respond to external stimuli. The rice-blast fungus Magnaporthe grisea forms specialized infection structures called appressoria in response to inductive surface cues. We isolated Magnaporthe RGS1 in a screen for mutants that form precocious appressoria on non-inductive surfaces. We report that a thigmotropic cue is necessary for initiating appressoria and for accumulating cAMP. Similar to an RGS1-deletion strain, magA(G187S) (RGS-insensitive Galpha(s)) and magA(Q208L) (GTPase-dead) mutants accumulated excessive cAMP and elaborated appressoria on non-inductive surfaces, suggesting that Rgs1 regulates MagA during pathogenesis. Rgs1 was also found to negatively regulate the Galpha(i) subunit MagB during asexual development. Deficiency of MAGB suppressed the hyper-conidiation defect in RGS1-deletion strain, whereas magB(G183S) and magB(Q204L) mutants produced more conidia, similar to the RGS1-deletion strain. Rgs1 physically interacted with GDP.AlF(4)(-)-activated forms of MagA, MagB and MagC (a Galpha(II) subunit). Thus, Rgs1 serves as a negative regulator of all Galpha subunits in Magnaporthe and controls important developmental events during asexual and pathogenic development.     

Functional Role of Amino-Terminal Serine16 and Serine27 of Gαz in Receptor and Effector Coupling

Abstract: The α subunit of G_z (α_z) harbors two N-terminal serine residues (at positions 16 and 27) that serve as protein kinase C-mediated phosphorylation sites. The cognate residues in the α subunit of G_t1 provide binding surfaces for the β₁ subunit. We used three serine-to-alanine mutants of α_z to investigate the functional importance of the two N-terminal serine residues. Wild-type or mutant α_z was transiently coexpressed with different receptors and adenylyl cyclase isozymes in human embryonic kidney 293 cells, and agonist-dependent regulation of cyclic AMP accumulation was examined in a setting where all endogenous α subunits of G_i were inactivated by pertussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of α_z to interact with δ-opioid, dopamine D₂, or adenosine A₁ receptors. Its capacity to inhibit endogenous and type VI adenylyl cyclases was also unaffected. Functional release of βγ subunits from the mutant α_z subunits was not impaired because they transduced βγ-mediated stimulation of type II adenylyl cyclase. Constitutively active mutants of all four α_z subunits were constructed by the introduction of a Q205L mutation. The activated mutants showed differential abilities to inhibit human choriogonadotropin-mediated cyclic AMP accumulation in luteinizing hormone receptor-transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor-independent inhibition of adenylyl cyclase by the GTPase-deficient mutant. Thus, replacement of the amino-terminal serine residues of α_z has no apparent effect on receptor-mediated responses, but these serine residues may be essential for ensuring transition of α_z into the active conformation.     

A novel strategy to engineer functional fluorescent inhibitory G-protein alpha subunits

Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein alpha subunits. We have designed G(i/o)alpha subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-protein-gated inwardly rectifying K(+) channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G(i/o) alpha subunits (G(i1)alpha, G(i2)alpha, G(i3)alpha, and G(oA)alpha) and a mutation introduced at Cys(-4) rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G(i/o)alpha protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G(i/o) alpha subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the Gbetagamma dimer by the mutant Galpha subunit. Moreover, each subunit was able to support A1-mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G(i3)alpha and G(oA)alpha alpha subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.     

New insights into the role of conserved, essential residues in the GTP binding/GTP hydrolytic cycle of large G proteins

The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the alpha subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of Galpha subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a Galpha chimera (designated alpha(T)*), comprised mainly of the alpha subunit of retinal transducin (alpha(T)) and the Switch III region from the alpha subunit of G(i1). As expected, both the alpha(T)*(R174C) and alpha(T)*(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and Gbetagamma. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and Gbetagamma to undergo GDP-[(35)S]GTPgammaS exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotide-bound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P(i) complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal Galpha subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.     

Delineation of the roles played by RasG and RasC in cAMP-dependent signal transduction during the early development of Dictyostelium discoideum

On starvation, the cellular slime mold Dictyostelium discoideum initiates a program of development leading to formation of multicellular structures. The initial cell aggregation requires chemotaxis to cyclic AMP (cAMP) and relay of the cAMP signal by the activation of adenylyl cyclase (ACA), and it has been shown previously that the Ras protein RasC is involved in both processes. Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG also has a role in early development. Both chemotaxis and ACA activation were reduced in the rasG- cells, but the effect on chemotaxis was more pronounced. When the responses of rasG- cells to cAMP were compared with the responses of rasC- and rasC- rasG- strains, generated in otherwise isogenic backgrounds, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Because the loss of either of the two Ras proteins alone did not result in a total loss of signal output down either of the branches of the cAMP signal-response pathway, there appears to be some overlap of function.     

Increased mitogenic responsiveness of Swiss 3T3 cells expressing constitutively active Gs alpha

Mutational replacement of glutamine-227 with a leucine residue in the GTP-binding domain of the alpha subunit of GS (Q227L alpha S) reduces its ability to hydrolyse GTP and causes constitutive activation of the mutant protein. Expression in Swiss 3T3 fibroblasts of Q227L alpha S caused markedly increased basal adenylyl cyclase activity, enhanced intracellular cyclic AMP (cAMP) accumulation and increased mitogenic sensitivity in response to forskolin and the potent phosphodiesterase inhibitor Ro 20-1724. These results support a role for cAMP in the regulation of cell proliferation, and suggest that alterations in a G protein can directly modify the ability of cells to respond mitogenically to extracellular factors.     

Galpha-mediated inhibition of developmental signal response

BACKGROUND: Seven-transmembrane receptor (7-TMR)-G protein networks are molecular sensors of extracellular signals in all eukarya. These pathways cycle through activated (sensitized) and inhibited (desensitized) states, and, while many of the molecular components for signal activation have been described, inhibitory mechanisms are not well characterized. In Dictyostelium, 7-TM cAMP receptors direct chemotaxis and development but also regulate the periodic synthesis of their own ligand, the chemoattractant/morphogen cAMP. We now demonstrate through loss-of-function/gain-of-function studies that the novel heterotrimeric Galpha9 protein subunit regulates an inhibitory pathway during early Dictyostelium development for the cAMP signal response.RESULTS: galpha9 null cells form more cAMP signaling centers, are more resistant to compounds that inhibit cAMP signaling, and complete aggregation sooner and at lower cell densities than wild-type cells. These phentoypes are consistent with the loss of an inhibitory signaling pathway during development of galpha9 null cells. Cells expressing constitutively activated Galpha9 are defective in cAMP signaling center formation and development at low cell density and display an increased sensitivity to cAMP signal inhibition that is characteristic of enhanced suppression of the cAMP signal response. Finally, we demonstrate that galpha9 null cells, which have been codeveloped with a majority of wild-type cells, primarily establish cAMP signaling centers and are able to non-autonomously direct wild-type cells to adopt a galpha9 null-like phenotype.CONCLUSIONS: We suggest that Galpha9 functions in an inhibitory-feedback pathway that regulates cAMP signaling center formation and propagation. Galpha9 may be part of the mechanism that regulates lateral signal inhibition or that modulates receptor desensitization.     

A point mutation to Galphai selectively blocks GoLoco motif binding: direct evidence for Galpha.GoLoco complexes in mitotic spindle dynamics

Heterotrimeric G-protein Galpha subunits and GoLoco motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Galphai) of Galpha subunits, and thus it is assumed that a Galphai.GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Galphai subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the alphaD-alphaE loop of the Galpha helical domain. This GoLoco-insensitivity ("GLi") mutation prevented Galphai1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Galpha subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Galphai subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gbetagamma dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Galphai subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Galphai subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Galphai.GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco motifs in receptor-mediated signal transduction.     

Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression.

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.     

Differential coupling of the extreme C-terminus of G protein alpha subunits to the G protein-coupled melatonin receptors

Melatonin receptors interact with pertussis toxin-sensitive G proteins to inhibit adenylate cyclase. However, the G protein coupling profiles of melatonin receptor subtypes have not been fully characterised and alternative G protein coupling is evident. The five C-terminal residues of Galpha subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Galphas chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Galpha subunits. The Galphas portion of the chimaeras confers the ability to activate adenylate cyclase leading to cyclic AMP production. Co-transfection of HEK293 cells expressing MT(1) or MT(2) melatonin receptors with Galphas chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Galpha C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Galpha chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Galphas chimaeras coupled. Recognition of the C-terminal five amino acids of the Galpha subunit is a requisite for coupling to a receptor, but it is not the sole determinant.     

Gbetagamma inhibits Galpha GTPase-activating proteins by inhibition of Galpha-GTP binding during stimulation by receptor

Gbetagamma subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, Gbetagamma subunits help anchor Galpha subunits to the plasma membrane, promote interaction of Galpha with receptors, stabilize the binding of GDP to Galpha to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. Gbetagamma subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-betas and RGS proteins, when assayed in solution under single turnover conditions. We show here that Gbetagamma subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires Gbetagamma in amounts approximately equimolar to Galpha, but GAP inhibition was observed with superstoichiometric Gbetagamma. The potency of inhibition varied with the GAP and the Galpha subunit, but half-maximal inhibition of the GAP activity of PLC-beta1 was observed with 5-10 nM Gbetagamma, which is at or below the concentrations of Gbetagamma needed for regulation of physiologically relevant effector proteins. The kinetics of GAP inhibition of both receptor-stimulated GTPase activity and single turnover, solution-based GAP assays suggested a competitive mechanism in which Gbetagamma competes with GAPs for binding to the activated, GTP-bound Galpha subunit. An N-terminal truncation mutant of PLC-beta1 that cannot be directly regulated by Gbetagamma remained sensitive to inhibition of its GAP activity, suggesting that the Gbetagamma binding site relevant for GAP inhibition is on the Galpha subunit rather than on the GAP. Using fluorescence resonance energy transfer between cyan or yellow fluorescent protein-labeled G protein subunits and Alexa532-labeled RGS4, we found that Gbetagamma directly competes with RGS4 for high-affinity binding to Galpha(i)-GDP-AlF4.     

Diverse chemotactic responses of Dictyostelium discoideum amoebae in the developing (temporal) and stationary (spatial) concentration gradients of folic acid,cAMP, Ca(2+) and Mg(2+)

The responses of Dictyostelium discoideum amoebae to developing (temporal) and stationary (spatial) gradients of folic acid, cAMP, Ca(2+), and Mg(2+) were studied using the methods of computer-aided image analysis. The results presented demonstrate that the new type of experimental chambers used for the observation of single cells moving within the investigated gradients of chemoattractants permit time lapse recording of single amoebae and determination of the trajectories of moving cells. It was found that, besides folic acid and cAMP (natural chemoattractants for Dictyostelium discoideum amoebae), also extracellular Ca(2+) and Mg(2+) are potent inducers of these cells' chemotaxis, and the amoebae of D. discoideum can respond to various chemoattractants differently. In the positively developing gradients of folic acid, cAMP, Ca(2+), and Mg(2+) oriented locomotion of amoebae directed towards the higher concentration of the tested chemoattractants was observed. However, in the negatively developing (temporal) and stationary linear (spatial) gradients, the univocal chemotaxis of amoebae was recorded only in the case of the Mg(2+) concentration gradient. This demonstrates that amoebae can respond to both developing and stationary gradients, depending upon the nature of the chemoattractant. We also investigated the effects of chosen inhibitors of signalling pathways upon chemotaxis of D. discoideum amoebae in the positively developing (temporal) gradients of tested chemoattractants. Verapamil was found to abolish the chemotaxis of amoebae only in the Ca(2+) gradients. Pertussis toxin suppressed the chemotactic response of cells in the gradients of folic acid and cAMP but did not prevent chemotaxis in those of Ca(2+) and Mg(2+), while quinacrine inhibited chemotaxis in the gradients of folic acid, cAMP, and Ca(2+) but only slightly affected chemotaxis in the Mg(2+) gradient. None of the tested inhibitors causes inhibition of cell random movement, when applied in isotropic solution. Also EDTA and EGTA up to 50 mM concentration did not inhibit locomotion of amoebae in control isotropic solutions.     

Regulation and function of G alpha protein subunits in Dictyostelium

We have examined the developmental regulation and function of two G alpha protein subunits, G alpha 1 and G alpha 2, from Dictyostelium. G alpha 1 is expressed in vegetative cells through aggregate stages while G alpha 2 is inducible by cAMP pulses and preferentially expressed in aggregation. Our results suggest that G alpha 2 encodes the G alpha protein subunit associated with the cAMP receptor and mediates all known receptor-activated intracellular signal transduction processes, including chemotaxis and gene regulation. G alpha 1 appears to function in both the cell cycle and development. Overexpression of G alpha 1 results in large, multinucleated cells that develop abnormally. The central role that these G alpha proteins play in signal transduction processes and in controlling Dictyostelium development is discussed.     

Gbetagamma-activated inwardly rectifying K(+) (GIRK) channel activation kinetics via Galphai and Galphao-coupled receptors are determined by Galpha-specific interdomain interactions that affect GDP release rates

Gbetagamma-activated inwardly rectifying K(+) (GIRK) channels have distinct gating properties when activated by receptors coupled specifically to Galpha(o) versus Galpha(i) subunit isoforms, with Galpha(o)-coupled currents having approximately 3-fold faster agonist-evoked activation kinetics. To identify the molecular determinants in Galpha subunits mediating these kinetic differences, chimeras were constructed using pertussis toxin (PTX)-insensitive Galpha(oA) and Galpha(i2) mutant subunits (Galpha(oA(C351G)) and Galpha(i2(C352G))) and examined in PTX-treated Xenopus oocytes expressing muscarinic m2 receptors and Kir3.1/3.2a channels. These experiments revealed that the alpha-helical N-terminal region (amino acids 1-161) and the switch regions of Galpha(i2) (amino acids 162-262) both partially contribute to slowing the GIRK activation time course when compared with the Galpha(oA(C351G))-coupled response. When present together, they fully reproduce Galpha(i2(C352G))-coupled GIRK kinetics. The Galpha(i2) C-terminal region (amino acids 263-355) had no significant effect on GIRK kinetics. Complementary responses were observed with chimeras substituting the Galpha(o) switch regions into the Galpha(i2(C352G)) subunit, which partially accelerated the GIRK activation rate. The Galpha(oA)/Galpha(i2) chimera results led us to examine an interaction between the alpha-helical domain and the Ras-like domain previously implicated in mediating a 4-fold slower in vitro basal GDP release rate in Galpha(i1) compared with Galpha(o). Mutations disrupting the interdomain contact in Galpha(i2(C352G)) at either the alphaD-alphaE loop (R145A) or the switch III loop (L233Q/A236H/E240T/M241T), significantly accelerated the GIRK activation kinetics consistent with the Galpha(i2) interdomain interface regulating receptor-catalyzed GDP release rates in vivo. We propose that differences in Galpha(i) versus Galpha(o)-coupled GIRK activation kinetics are due to intrinsic differences in receptor-catalyzed GDP release that rate-limit Gbetagamma production and is attributed to heterogeneity in Galpha(i) and Galpha(o) interdomain contacts.