LET-99 opposes Galpha/GPR signaling to generate asymmetry for spindle positioning in response to PAR and MES-1/SRC-1 signaling

G-protein signaling plays important roles in asymmetric cell division. In C. elegans embryos, homologs of receptor-independent G protein activators, GPR-1 and GPR-2 (GPR-1/2), function together with Galpha (GOA-1 and GPA-16) to generate asymmetric spindle pole elongation during divisions in the P lineage. Although Galpha is uniformly localized at the cell cortex, the cortical localization of GPR-1/2 is asymmetric in dividing P cells. In this report, we show that the asymmetry of GPR-1/2 localization depends on PAR-3 and its downstream intermediate LET-99. Furthermore, in addition to its involvement in spindle elongation, Galpha is required for the intrinsically programmed nuclear rotation event that orients the spindle in the one-cell. LET-99 functions antagonistically to the Galpha/GPR-1/2 signaling pathway, providing an explanation for how Galpha-dependent force is regulated asymmetrically by PAR polarity cues during both nuclear rotation and anaphase spindle elongation. In addition, Galpha and LET-99 are required for spindle orientation during the extrinsically polarized division of EMS cells. In this cell, both GPR-1/2 and LET-99 are asymmetrically localized in response to the MES-1/SRC-1 signaling pathway. Their localization patterns at the EMS/P2 cell boundary are complementary, suggesting that LET-99 and Galpha/GPR-1/2 signaling function in opposite ways during this cell division as well. These results provide insight into how polarity cues are transmitted into specific spindle positions in both extrinsic and intrinsic pathways of asymmetric cell division.     

Control of embryonic spindle positioning and Galpha activity by C. elegans RIC-8

Asymmetric spindle positioning is of fundamental importance for generating cell diversity during development. In the C. elegans 1 cell embryo, spindle positioning has been shown to depend on heterotrimeric G protein signaling. Two Galpha subunits, GOA-1 and GPA-16 (hereafter Galpha), and receptor independent activators of G protein signaling GPR-1 and GPR-2 (GPR-1/2) are required for proper regulation of spindle positioning . However, it remains unclear whether Galpha regulates spindle positioning in its GDP or GTP bound form. Here, we investigate the role of RIC-8 in this pathway. RIC-8 was genetically shown to act in concert with goa-1 to regulate centrosome movements in C. elegans . Interestingly, mammalian RIC-8 was recently found to behave as a GEF for Galpha subunits in vitro . We show that reduction of function of ric-8 results in a 1 cell embryo phenotype very similar to the phenotype of embryos depleted of Galpha. RIC-8 is able to directly bind to GOA-1, preferentially to GOA-1-GDP, consistent with a GEF role. RIC-8 is localized at the embryo cortex, and its activity is essential for the asymmetric localization of GPR-1/2. We suggest that RIC-8 directly modulates Galpha activity and that Galpha-GTP is the signaling molecule regulating spindle positioning in the early embryo.     

Cortical localization of the Galpha protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division

Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C. elegans embryos, the Galpha proteins GOA-1 and GPA-16 act in a partially redundant manner to generate pulling forces along astral microtubules. Previous work focused primarily on GOA-1, whereas the mechanisms by which GPA-16 participates in this process are not well understood. Here, we report that GPA-16 is present predominantly at the cortex of one-cell stage embryos. Using co-immunoprecipitation and surface plasmon resonance binding assays, we find that GPA-16 associates with RIC-8 and GPR-1/2, two proteins known to be required for pulling force generation. Using spindle severing as an assay for pulling forces, we demonstrate that inactivation of the Gbeta protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two Galpha proteins can activate the same pathway and that their dual presence is normally needed to counter Gbetagamma. Using nucleotide exchange assays, we establish that whereas GPR-1/2 acts as a guanine nucleotide dissociation inhibitor (GDI) for GPA-16, as it does for GOA-1, RIC-8 does not exhibit guanine nucleotide exchange factor (GEF) activity towards GPA-16, in contrast to its effect on GOA-1. We establish in addition that RIC-8 is required for cortical localization of GPA-16, whereas it is not required for that of GOA-1. Our analysis demonstrates that this requirement toward GPA-16 is distinct from the known function of RIC-8 in enabling interaction between Galpha proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.     

A casein kinase 1 and PAR proteins regulate asymmetry of a PIP(2) synthesis enzyme for asymmetric spindle positioning

Spindle positioning is an essential feature of asymmetric cell division. The conserved PAR proteins together with heterotrimeric G proteins control spindle positioning in animal cells, but how these are linked is not known. In C. elegans, PAR protein activity leads to asymmetric spindle placement through cortical asymmetry of Galpha regulators GPR-1/2. Here, we establish that the casein kinase 1 gamma CSNK-1 and a PIP(2) synthesis enzyme (PPK-1) transduce PAR polarity to asymmetric Galpha regulation. PPK-1 is posteriorly enriched in the one-celled embryo through PAR and CSNK-1 activities. Loss of CSNK-1 causes uniformly high PPK-1 levels, high symmetric cortical levels of GPR-1/2 and LIN-5, and increased spindle pulling forces. In contrast, knockdown of ppk-1 leads to low GPR-1/2 levels and decreased spindle forces. Furthermore, loss of CSNK-1 leads to increased levels of PIP(2). We propose that asymmetric generation of PIP(2) by PPK-1 directs the posterior enrichment of GPR-1/2 and LIN-5, leading to posterior spindle displacement.     

RIC-8 is required for GPR-1/2-dependent Galpha function during asymmetric division of C. elegans embryos

Heterotrimeric G proteins are crucial for asymmetric cell division, but the mechanisms of signal activation remain poorly understood. Here, we establish that the evolutionarily conserved protein RIC-8 is required for proper asymmetric division of one-cell stage C. elegans embryos. Spindle severing experiments demonstrate that RIC-8 is required for generation of substantial pulling forces on astral microtubules. RIC-8 physically interacts with GOA-1 and GPA-16, two Galpha subunits that act in a partially redundant manner in one-cell stage embryos. RIC-8 preferentially binds to GDP bound GOA-1 and is a guanine nucleotide exchange factor (GEF) for GOA-1. Our analysis suggests that RIC-8 acts before the GoLoco protein GPR-1/2 in the sequence of events leading to Galpha activation. Furthermore, coimmunoprecipitation and in vivo epistasis demonstrate that inactivation of the Gbeta subunit GPB-1 alleviates the need for RIC-8 in one-cell stage embryos. Our findings suggest a mechanism in which RIC-8 favors generation of Galpha free from Gbetagamma and enables GPR-1/2 to mediate asymmetric cell division.     

Distinct roles for Galpha and Gbetagamma in regulating spindle position and orientation in Caenorhabditis elegans embryos

Correct placement and orientation of the mitotic spindle is essential for segregation of localized components and positioning of daughter cells. Although these processes are important in many cells, few factors that regulate spindle placement are known. Previous work has shown that GPB-1, the Gbeta subunit of a heterotrimeric G protein, is required for orientation of early cell division axes in C. elegans embryos. Here we show that GOA-1 (a Galphao) and the related GPA-16 are the functionally redundant Galpha subunits and that GPC-2 is the relevant Ggamma subunit that is required for spindle orientation in the early embryo. We show that Galpha and Gbetagamma are involved in controlling distinct microtubule-dependent processes. Gbetagamma is important in regulating migration of the centrosome around the nucleus and hence in orientating the mitotic spindle. Galpha is required for asymmetric spindle positioning in the one-celled embryo.     

LET-99, GOA-1/GPA-16, and GPR-1/2 are required for aster-positioned cytokinesis

At anaphase, the mitotic spindle positions the cytokinesis furrow [1]. Two populations of spindle microtubules are implicated in cytokinesis: radial microtubule arrays called asters and bundled nonkinetochore microtubules called the spindle midzone [2-4]. In C. elegans embryos, these two populations of microtubules provide two consecutive signals that position the cytokinesis furrow: The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone [5]. Evidence for two cytokinesis signals came from the identification of molecules that block midzone-positioned cytokinesis [5-7]. However, no molecules that are only required for, and thus define, the molecular pathway of aster-positioned cytokinesis have been identified. With RNAi screening, we identify LET-99 and the heterotrimeric G proteins GOA-1/GPA-16 and their regulator GPR-1/2 [10-12] in aster-positioned cytokinesis. By using mechanical spindle displacement, we show that the anaphase spindle positions cortical LET-99, at the site of the presumptive cytokinesis furrow. LET-99 enrichment at the furrow depends on the G proteins. GPR-1 is locally reduced at the site of cytokinesis-furrow formation by LET-99, which prevents accumulation of GPR-1 at this site. We conclude that LET-99 and the G proteins define a molecular pathway required for aster-positioned cytokinesis.     

Interaction of activator of G-protein signaling 3 (AGS3) with LKB1, a serine/threonine kinase involved in cell polarity and cell cycle progression: phosphorylation of the G-protein regulatory (GPR) motif as a regulatory mechanism for the interaction of GPR motifs with Gi alpha

Activator of G-protein signaling 3 (AGS3) has a modular domain structure consisting of seven tetratricopeptide repeats (TPRs) and four G-protein regulatory (GPR) motifs. Each GPR motif binds to the alpha subunit of Gi/Go (Gialpha > Goalpha) stabilizing the GDP-bound conformation of Galpha and apparently competing with Gbetagamma for GalphaGDP binding. As an initial approach to identify regulatory mechanisms for AGS3-G-protein interactions, a yeast two-hybrid screen was initiated using the TPR and linker region of AGS3 as bait. This screen identified the serine/threonine kinase LKB1, which is involved in the regulation of cell cycle progression and polarity. Protein interaction assays in mammalian systems using transfected cells or brain lysate indicated the regulated formation of a protein complex consisting of LKB1, AGS3, and G-proteins. The interaction between AGS3 and LKB1 was also observed with orthologous proteins in Drosophila where both proteins are involved in cell polarity. LKB1 immunoprecipitates from COS7 cells transfected with LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain. GPR domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phosphate moiety within a consensus GPR motif reduced the ability of the peptide to interact with G-proteins. These data suggest that phosphorylation of GPR domains may be a general mechanism regulating the interaction of GPR-containing proteins with G-proteins. Such a mechanism may be of particular note in regard to localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur independently of a typical G-protein-coupled receptor.     

Embryonic handedness choice in C. elegans involves the Galpha protein GPA-16

The mechanism by which polarity of the left-right (LR) axis is initially established with the correct handedness is not understood for any embryo. C. elegans embryos exhibit LR asymmetry with an invariant handedness that is first apparent at the six-cell stage and persists throughout development. We show here that a strong loss-of-function mutation in a gene originally designated spn-1 affects early spindle orientations and results in near randomization of handedness choice. This mutation interacts genetically with mutations in three par genes that encode localized cortical components. We show that the spn-1 gene encodes the Galpha protein GPA-16, which appears to be required for centrosomal association of a Gbeta protein. We will henceforth refer to this gene as gpa-16. These results demonstrate for the first time involvement of heterotrimeric G proteins in establishment of embryonic LR asymmetry and suggest how they might act.     

Influence of cytosolic AGS3 on receptor--G protein coupling

Activator of G protein signaling 3 (AGS3) activates the Gbetagamma mating pathway in yeast in a manner that is independent of heptahelical receptors. It competes with Gbetagamma subunits to bind GDP-bound Gi/o(alpha) subunits via four repeated G protein regulatory (GPR) domains in the carboxyl-terminal half of the molecule. However, little is known about the functional role of AGS3 in cellular signaling. Here the effect of AGS3 on receptor-G protein coupling was examined in an Sf9 cell membrane-based reconstitution system. A GST-AGS3-GPR fusion protein containing the four individual AGS3-GPR domains inhibits receptor coupling to Galpha subunits as effectively as native AGS3 and more effectively than GST fusion proteins containing the individual AGS3-GPR domains. While none of the GPR domains distinguished among the three G(i)alpha subunits, both individual and full-length GPR domains interacted more weakly with G(o)alpha than with G(i)alpha. Cytosolic AGS3, but not membrane-associated AGS3, can interact with G(i)alpha subunits and disrupt their receptor coupling. Immunoblotting studies reveal that cytosolic AGS3 can remove G(i)alpha subunits from the membrane and sequester G(i)alpha subunits in the cytosol. These findings suggest that AGS3 may downregulate heterotrimeric G protein signaling by interfering with receptor coupling.     

Control of nuclear centration in the C. elegans zygote by receptor-independent Galpha signaling and myosin II

Mitotic spindle positioning in the Caenorhabditis elegans zygote involves microtubule-dependent pulling forces applied to centrosomes. In this study, we investigate the role of actomyosin in centration, the movement of the nucleus-centrosome complex (NCC) to the cell center. We find that the rate of wild-type centration depends equally on the nonmuscle myosin II NMY-2 and the Galpha proteins GOA-1/GPA-16. In centration- defective let-99(-) mutant zygotes, GOA-1/GPA-16 and NMY-2 act abnormally to oppose centration. This suggests that LET-99 determines the direction of a force on the NCC that is promoted by Galpha signaling and actomyosin. During wild-type centration, NMY-2-GFP aggregates anterior to the NCC tend to move further anterior, suggesting that actomyosin contraction could pull the NCC. In GOA-1/GPA-16-depleted zygotes, NMY-2 aggregate displacement is reduced and largely randomized, whereas in a let-99(-) mutant, NMY-2 aggregates tend to make large posterior displacements. These results suggest that Galpha signaling and LET-99 control centration by regulating polarized actomyosin contraction.     

Signaling by a non-dissociated complex of G protein βγ and α subunits stimulated by a receptor-independent activator of G protein signaling, AGS8

Accumulating evidence suggests that heterotrimeric G protein activation may not require G protein subunit dissociation. Results presented here provide evidence for a subunit dissociation-independent mechanism for G protein activation by a receptor-independent activator of G protein signaling, AGS8. AGS8 is a member of the AGS group III family of AGS proteins thought to activate G protein signaling primarily through interactions with Gbetagamma subunits. Results are presented demonstrating that AGS8 binds to the effector and alpha subunit binding "hot spot" on Gbetagamma yet does not interfere with Galpha subunit binding to Gbetagamma or phospholipase C beta2 activation. AGS8 stimulates activation of phospholipase C beta2 by heterotrimeric Galphabetagamma and forms a quaternary complex with Galpha(i1), Gbeta(1)gamma(2), and phospholipase C beta2. AGS8 rescued phospholipase C beta binding and regulation by an inactive beta subunit with a mutation in the hot spot (beta(1)(W99A)gamma(2)) that normally prevents binding and activation of phospholipase C beta2. This demonstrates that, in the presence of AGS8, the hot spot is not used for Gbetagamma interactions with phospholipase C beta2. Mutation of an alternate binding site for phospholipase C beta2 in the amino-terminal coiled-coil region of Gbetagamma prevented AGS8-dependent phospholipase C binding and activation. These data implicate a mechanism for AGS8, and potentially other Gbetagamma binding proteins, for directing Gbetagamma signaling through alternative effector activation sites on Gbetagamma in the absence of subunit dissociation.     

Dynamic localization of LIN-5 and GPR-1/2 to cortical force generation domains during spindle positioning

G protein signaling pathways regulate mitotic spindle positioning during cell division in many systems. In Caenorhabditis elegans embryos, Gα subunits act with the positive regulators GPR-1/2 and LIN-5 to generate cortical pulling forces for posterior spindle displacement during the first asymmetric division. GPR-1/2 are asymmetrically localized at the posterior cortex by PAR polarity cues at this time. Here we show that LIN-5 colocalizes with GPR-1/2 in one-cell embryos during spindle displacement. Significantly, we also find that LIN-5 and GPR-1/2 are localized to the opposite, anterior cortex in a polarity-dependent manner during the nuclear centration and rotation movements that orient the forming spindle onto the polarity axis. The depletion of LIN-5 or GPR-1/2 results in decreased centration and rotation rates, indicating a role in force generation at this stage. The localization of LIN-5 and GPR-1/2 is largely interdependent and requires Gα. Further, LIN-5 immunoprecipitates with Gα in vivo, and this association is GPR-1/2 dependent. These results suggest that a complex of Gα/GPR-1/2/LIN-5 is asymmetrically localized in response to polarity cues, and this may be the active signaling complex that transmits asymmetries to the force generation machinery during both nuclear rotation and spindle displacement.     

Structural determinants underlying the temperature-sensitive nature of a Galpha mutant in asymmetric cell division of Caenorhabditis elegans

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Galpha(o)) and GPA-16 (Galpha(i)) are involved in generating forces that pull on astral microtubules and position the spindle asymmetrically. GPA-16 function has been analyzed in vivo owing notably to a temperature-sensitive allele gpa-16(it143), which, at the restrictive temperature, results in spindle orientation defects in early embryos. Here we identify the structural basis of gpa-16(it143), which encodes a point mutation (G202D) in the switch II region of GPA-16. Using Galpha(i1)(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Galpha. At the permissive temperature, the mutant Galpha was stable upon GTP binding, but switch II rearrangement was compromised, as were activation state-selective interactions with regulators involved in ACD, including GoLoco motifs, RGS proteins, and RIC-8. We solved the crystal structure of the mutant Galpha bound to GDP, which indicates a unique switch II conformation as well as steric constraints that suggest activated GPA-16(it143) is destabilized relative to wild type. Spindle severing in gpa-16(it143) embryos revealed that pulling forces are symmetric and markedly diminished at the restrictive temperature. Interestingly, pulling forces are asymmetric and generally similar in magnitude to wild type at the permissive temperature despite defects in the structure of GPA-16(it143). These normal pulling forces in gpa-16(it143) embryos at the permissive temperature were attributable to GOA-1 function, underscoring a complex interplay of Galpha subunit function in ACD.     

PAR-3 and PAR-1 inhibit LET-99 localization to generate a cortical band important for spindle positioning in Caenorhabditis elegans embryos

The conserved PAR proteins are localized in asymmetric cortical domains and are required for the polarized localization of cell fate determinants in many organisms. In Caenorhabditis elegans embryos, LET-99 and G protein signaling act downstream of the PARs to regulate spindle positioning and ensure asymmetric division. PAR-3 and PAR-2 localize LET-99 to a posterior cortical band through an unknown mechanism. Here we report that LET-99 asymmetry depends on cortically localized PAR-1 and PAR-4 but not on cytoplasmic polarity effectors. In par-1 and par-4 embryos, LET-99 accumulates at the entire posterior cortex, but remains at low levels at the anterior cortex occupied by PAR-3. Further, PAR-3 and PAR-1 have graded cortical distributions with the highest levels at the anterior and posterior poles, respectively, and the lowest levels of these proteins correlate with high LET-99 accumulation. These results suggest that PAR-3 and PAR-1 inhibit the localization of LET-99 to generate a band pattern. In addition, PAR-1 kinase activity is required for the inhibition of LET-99 localization, and PAR-1 associates with LET-99. Finally, examination of par-1 embryos suggests that the banded pattern of LET-99 is critical for normal posterior spindle displacement and to prevent spindle misorientation caused by cell shape constraints.     

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.     

Activation of heterotrimeric G-protein signaling by a ras-related protein. Implications for signal integration

Utilizing a functional screen in the yeast Saccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion. One of the identified activators, termed AGS1 (for activator of G-protein signaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily. Expression of AGS1 in yeast and in mammalian cells results in specific activation of Galpha(i)/Galpha(o) heterotrimeric signaling pathways. In addition, the in vivo and in vitro properties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for Galpha(i)/Galpha(o). AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways.     

PAR proteins regulate microtubule dynamics at the cell cortex in C. elegans

BACKGROUND: The PAR proteins are known to be localized asymmetrically in polarized C. elegans, Drosophila, and human cells and to participate in several cellular processes, including asymmetric cell division and spindle orientation. Although astral microtubules are known to play roles in these processes, their behavior during these events remains poorly understood. RESULTS: We have developed a method that makes it possible to examine the residence time of individual astral microtubules at the cell cortex of developing embryos. Using this method, we found that microtubules are more dynamic at the posterior cortex of the C. elegans embryo compared to the anterior cortex during spindle displacement. We further observed that this asymmetry depends on the PAR-3 protein and heterotrimeric G protein signaling, and that the PAR-2 protein affects microtubule dynamics by restricting PAR-3 activity to the anterior of the embryo. CONCLUSIONS: These results indicate that PAR proteins function to regulate microtubule dynamics at the cortex during microtubule-dependent cellular processes.     

Axis determination in C. elegans: initiating and transducing polarity.

The anterior-posterior axis in Caenorhabditis elegans is determined by the sperm and leads to the asymmetric localisation of PAR (partitioning-defective) proteins, which are critical for polarity. New findings demonstrate that sperm asters play a critical role and suggest models for how PAR asymmetry is established. In addition, studies of blastomere fate determination and heterotrimeric G proteins have started to uncover how initial polarity may be translated into the asymmetric distribution of maternal proteins and the control of spindle position.     

Receptor- and nucleotide exchange-independent mechanisms for promoting G protein subunit dissociation

Mechanisms for heterotrimeric G protein activation that do not rely on G protein coupled receptor activation are becoming increasingly apparent. We recently identified beta gamma subunit-binding peptides that we proposed bound to a "hot spot" on beta gamma subunits, stimulating G protein dissociation without stimulating nucleotide exchange and activating G protein signaling in intact cells. AGS3, a member of the activators of G protein signaling family of proteins, also activates G protein signaling in a nucleotide exchange-independent manner, and AGS3 homologues are involved in asymmetric cell division during development. Here we demonstrate that a consensus G protein regulatory (GPR) peptide from AGS3 and related proteins is sufficient to induce G protein subunit dissociation and that both the GPR and hot spot-binding peptides promote dissociation to extents comparable with a known G protein activator, AMF. Peptides derived from adenylyl cyclase 2 and GRK2 prevented formation of the heterotrimeric complex but did not alter the rate of alpha subunit dissociation from beta gamma subunits. These data indicate that these nucleotide exchange-independent G protein activator peptides do not simply compete for alpha interactions with beta gamma subunits, but actively promote subunit dissociation. Thus, we propose two novel mechanisms for nucleotide exchange independent activation of G protein signaling, one that involves conformational changes in the alpha subunit and one that involves conformational changes in the beta gamma subunits.