Geranylgeranylation signals to the Hippo pathway for breast cancer cell proliferation and migration.

Protein geranylgeranylation (GGylation) is an important biochemical process for many cellular signaling molecules. Previous studies have shown that GGylation is essential for cell survival in many types of cancer. However, the molecular mechanism mediating the cell survival effect remains elusive. In this report, we show that the Hippo pathway mediates GGylation-dependent cell proliferation and migration in breast cancer cells. Blockade of GGylation enhanced phosphorylation of Mst1/2 and Lats1, and inhibited YAP and TAZ activity and the Hippo-YAP/TAZ pathway-dependent transcription. The effect of GGylation blockade on inhibition of breast cancer cell proliferation and migration is dependent on the Hippo-YAP/TAZ signaling, in which YAP appears to regulate cell proliferation and TAZ to regulate cell migration. Furthermore, GGylation-dependent cell proliferation is correlated with the activity of YAP/TAZ in breast cancer cells. Finally, Gγ and RhoA are the GGylated proteins that may transduce GGylation signals to the Hippo-YAP/TAZ pathway. Taken together, our studies have demonstrated that the Hippo-YAP/TAZ pathway is essential for GGylation-dependent cancer cell proliferation and migration.

Differential dependence of the D1 and D5 dopamine receptors on the G protein gamma 7 subunit for activation of adenylylcyclase.

The D(1) dopamine receptor, G protein gamma(7) subunit, and adenylylcyclase are selectively expressed in the striatum, suggesting their potential interaction in a common signaling pathway. To evaluate this possibility, a ribozyme strategy was used to suppress the expression of the G protein gamma(7) subunit in HEK 293 cells stably expressing the human D(1) dopamine receptor. Prior in vitro analysis revealed that the gamma(7) ribozyme possessed cleavage activity directed exclusively toward the gamma(7) RNA transcript (Wang, Q., Mullah, B., Hansen, C., Asundi, J., and Robishaw, J. D. (1997) J. Biol. Chem. 272, 26040-26048). In vivo analysis of cells transfected with the gamma(7) ribozyme showed a specific reduction in the expression of the gamma(7) protein. Coincident with the loss of the gamma(7) protein, there was a noticeable reduction in the expression of the beta(1) protein, confirming their interaction in these cells. Finally, functional analysis of ribozyme-mediated suppression of the beta(1) and gamma(7) proteins revealed a significant attenuation of SKF81297-stimulated adenylylcyclase activity in D(1) dopamine receptor-expressing cells. By contrast, ribozyme-mediated suppression of the beta(1) and gamma(7) proteins showed no reduction of SKF81297-stimulated adenylylcyclase activity in D(5) dopamine receptor-expressing cells. Taken together, these data indicate that the structurally related D(1) and D(5) dopamine receptor subtypes utilize G proteins composed of distinct betagamma subunits to stimulate adenylylcyclase in HEK 293 cells. Underscoring the physiological relevance of these findings, single cell reverse transcriptase-polymerase chain reaction analysis revealed that the D(1) dopamine receptor and the G protein gamma(7) subunit are coordinately expressed in substance P containing neurons in rat striatum, suggesting that the G protein gamma(7) subunit may be a new target for drugs to selectively alter dopaminergic signaling within the brain.

Heterotrimeric G-protein betagamma-dimers in growth and differentiation.

Heterotrimeric G-proteins are components of the signal transduction pathways for the soluble and cell-contact signals that regulate normal growth and differentiation. There is now a greater appreciation of the role of the Gbetagamma-dimer in the regulation of a variety of intracellular effectors, including ion channels, adenylyl cyclase, and phospholipase Cbeta. In many cases, Gbetagamma-dimers are required for the activation of mitogen activated protein kinase (MAPK) pathways that promote cellular proliferation, although the underlying mechanisms have yet to be fully elucidated. Activation of phosphotidylinositol-3-kinase (PI3K) is a critical step in the intracellular transduction of survival signals. Gbetagamma-dimers directly activate PI3Kgamma as well as the more widely distributed PI3Kbeta. The activation of PI3Kgamma by Gbetagamma-dimers likely involves direct binding of specific Gbetagamma-dimers to both subunits of PI3Kgamma. Thus, Gbetagamma-dimers transmit signals from numerous receptors to a variety of intracellular effectors in distinct cellular contexts. Five distinct Gbeta-subunits and 12 distinct Ggamma-subunits have been identified. New experimental approaches are needed to elucidate the specific roles of individual Gbetagamma-dimers in the pathways that transduce signals for proliferation and survival.

A major locus for myoclonus-dystonia maps to chromosome 7q in eight families.

Myoclonus-dystonia (M-D) is an autosomal dominant disorder characterized by myoclonic and dystonic muscle contractions that are often responsive to alcohol. The dopamine D2 receptor gene (DRD2) on chromosome 11q has been implicated in one family with this syndrome, and linkage to a 28-cM region on 7q has been reported in another. We performed genetic studies, using eight additional families with M-D, to assess these two loci. No evidence for linkage was found for 11q markers. However, all eight of these families showed linkage to chromosome 7 markers, with a combined multipoint LOD score of 11.71. Recombination events in the families define the disease gene within a 14-cM interval flanked by D7S2212 and D7S821. These data provide evidence for a major locus for M-D on chromosome 7q21.

Human G protein gamma(11) and gamma(14) subtypes define a new functional subclass.

The mammalian gamma subunit family consists of a minimum of 12 members. Analysis of the amino acid sequence conservation suggests that the gamma subunit family can be divided into three distinct subclasses. The division of the gamma subunit family into these classes is based not only on amino acid homology, but also to some extent on functional similarities. In the present study, two new members of the gamma subunit family, the gamma(11) and gamma(14) subunits, are identified and characterized in terms of their expression and function. The gamma(11) and gamma(14) subunits are most closely related to the gamma(1) subunit and share similar biochemical properties, suggesting their inclusion in class I. However, despite their close phylogenetic relationship and similar biochemical properties, the gamma(1), gamma(11), and gamma(14) subunits exhibit very distinct expression patterns, suggesting that class I should be further subdivided and that the signaling functions of each subgroup are distinct. In this regard, the gamma(11) and gamma(14) subunits represent a new subgroup of farnesylated gamma subunits that are expressed outside the retina and have functions other than phototransduction.

Constitutively active mutants of the alpha(1a)- and the alpha(1b)-adrenergic receptor subtypes reveal coupling to different signaling pathways and physiological responses in rat cardiac myocytes.

Activation of alpha(1)-adrenergic receptors influences both the contractile activity and the growth potential of cardiac myocytes. However, the signaling pathways linking activation of specific alpha(1)-adrenergic receptor (AR) subtypes to these physiological responses remain controversial. In the present study, a molecular approach was used to identify conclusively the signaling pathways activated in response to the individual alpha(1A)- and alpha(1B)-AR subtypes in cardiac myocytes. For this purpose, a mutant alpha(1a)-AR subtype (alpha(1a)-S(290/293)-AR) was constructed based on analogy to the previously described constitutively active mutant alpha(1b)-AR subtype (alpha(1b)-S(288-294)-AR). The mutant alpha(1a)-S(290/293)-AR subtype displayed constitutive activity based on four criteria. To introduce the constitutively active alpha(1)-AR subtypes into cardiac myocytes, recombinant Sindbis viruses encoding either the alpha(1a)-S(290/293)-AR or alpha(1b)-S(288-294)-AR subtype were used to infect the whole cell population with >90% efficiency, thereby allowing the biochemical activities of the various signaling pathways to be measured. When expressed at comparable levels, the alpha(1a)-S(290/293)-AR subtype exhibited a significantly elevated basal level as well as agonist-stimulated level of inositol phosphate accumulation, coincident with activation of atrial natriuretic factor-luciferase gene expression. By contrast, the alpha(1b)-S(288-294)-AR subtype displayed a markedly increased serum response element-luciferase gene expression but no activation of atrial natriuretic factor-luciferase gene expression. Taken together, this study provides the first molecular evidence for coupling of the alpha(1a)-AR and the alpha(1b)-AR subtypes to different signaling pathways in cardiac myocytes.

Alpha-1 adrenergic signaling in a cardiac murine atrial myocyte (HL-1) cell line.

Activation of alpha-1 adrenergic receptors in the heart has been shown to result in increased contractile activity, cardiac fetal gene re-expression, and myocyte hypertrophy. Three alpha-1 adrenergic receptors have been identified through molecular cloning. Due to the limited selectivities of the currently available alpha-1 adrenergic receptor antagonists, the signaling pathways activated by specific subtypes in the heart remain unresolved. To resolve this dilemma, we have used a molecular approach to identify the signaling pathways and downstream genes that are engaged in response to activation of individual alpha-1 adrenergic subtypes in cardiac cells. We have transfected constitutively active alpha-1 adrenergic receptors (alpha1a-S290/293-AR [1] or the alpha1b-S288/294-AR [2]) subtypes into the cardiac murine myocyte cell line (HL-1) and studied the signal transduction pathway(s) and cardiac gene(s) activated by them. In this study, we demonstrate that the alpha1a-S290/293 -AR [1] subtype preferentially couples to cardiac-specific atrial natriuretic factor (ANF) gene expression, while the alpha1b-S288/294-AR preferentially couples to activation of mitogen-activated protein kinase (MAPK), Ets-like transcription factor-1 (Elk1) and serum response element (SRE) signaling pathways. Endogenous alpha-1 adrenergic receptors are expressed, and stimulate phosphatidylinositol-hydrolysis upon activation with the alpha-1 agonist, phenylephrine.

Ribozyme approach identifies a functional association between the G protein beta1gamma7 subunits in the beta-adrenergic receptor signaling pathway.

The complex role that the heterotrimeric G proteins play in signaling pathways has become increasingly apparent with the cloning of countless numbers of receptors, G proteins, and effectors. However, in most cases, the specific combinations of alpha and betagamma subunits comprising the G proteins that participate in the most common signaling pathways, such as beta-adrenergic regulation of adenylyl cyclase activity, are not known. The extent of this problem is evident in the fact that the identities of the betagamma subunits that combine with the alpha subunit of Gs are only now being elucidated almost 20 years after its initial purification. In a previous study, we described the first use of a ribozyme strategy to suppress specifically the expression of the gamma7 subunit of the G proteins, thereby identifying a specific role of this protein in coupling the beta-adrenergic receptor to stimulation of adenylyl cyclase activity in HEK 293 cells. In the present study, we explored the potential utility of a ribozyme approach directed against the gamma7 subunit to identify functional associations with a particular beta and alphas subunit of the G protein in this signaling pathway. Accordingly, HEK 293 cells were transfected with a ribozyme directed against the gamma7 subunit, and the effects of this manipulation on levels of the beta and alphas subunits were determined by immunoblot analysis. Among the five beta alphas subunits detected in these cells, only the beta1 subunit was coordinately reduced following treatment with the ribozyme directed against the gamma7 subunit, thereby demonstrating a functional association between the beta1 and gamma7 subunits. The mechanism for coordinate suppression of the beta1 subunit was due to a striking change in the half-life of the beta1 monomer versus the beta1 heterodimer complexed with the gamma7 subunit. Neither the 52- nor 45-kDa subunits were suppressed following treatment with the ribozyme directed against the gamma7 subunit, thereby providing insights into the assembly of the Gs heterotrimer. Taken together, these data show the utility of a ribozyme approach to identify the role of not only the gamma subunits but also the beta subunits of the G proteins in signaling pathways.

The alpha2A-adrenergic receptor discriminates between Gi heterotrimers of different betagamma subunit composition in Sf9 insect cell membranes.

In view of the expanding roles of the betagamma subunits of the G proteins in signaling, the possibility was raised that the rich diversity of betagamma subunit combinations might contribute to the specificity of signaling at the level of the receptor. To test this possibility, Sf9 cell membranes expressing the recombinant alpha2A-adrenergic receptor were used to assess the contribution of the betagamma subunit composition. Reconstituted coupling between the receptor and heterotrimeric Gi protein was assayed by high affinity, guanine nucleotide-sensitive binding of the alpha2-adrenergic agonist, [3H]UK-14,304. Supporting this hypothesis, the present study showed clear differences in the abilities of the various betagamma dimers, including those containing the beta3 subtype and the newly described gamma4, gamma10, and gamma11 subtypes, to promote interaction of the same alphai subunit with the alpha2A-adrenergic receptor.

Muscarinic K+ channel in the heart. Modal regulation by G protein beta gamma subunits.

The membrane-delimited activation of muscarinic K+ channels by G protein beta gamma subunits plays a prominent role in the inhibitory synaptic transmission in the heart. These channels are thought to be heterotetramers comprised of two homologous subunits, GIRK1 and CIR, both members of the family of inwardly rectifying K+ channels. Here, we demonstrate that muscarinic K+ channels in neonatal rat atrial myocytes exhibit four distinct gating modes. In intact myocytes, after muscarinic receptor activation, the different gating modes were distinguished by differences in both the frequency of channel opening and the mean open time of the channel, which accounted for a 76-fold increase in channel open probability from mode 1 to mode 4. Because of the tetrameric architecture of the channel, the hypothesis that each of the four gating modes reflects binding of a different number of Gbeta gamma subunits to the channel was tested, using recombinant Gbeta1 gamma5. Gbeta1 gamma5 was able to control the equilibrium between the four gating modes of the channel in a manner consistent with binding of Gbeta gamma to four equivalent and independent sites in the protein complex. Surprisingly, however, Gbeta1 gamma5 lacked the ability to stabilize the long open state of the channel that is responsible for the augmentation of the mean open time in modes 3 and 4 after muscarinic receptor stimulation. The modal regulation of muscarinic K+ channel gating by Gbeta gamma provides the atrial cells with at least two major advantages: the ability to filter out small inputs from multiple membrane receptors and yet the ability to create the gradients of information necessary to control the heart rate with great precision.

Ribozyme-mediated suppression of the G protein gamma7 subunit suggests a role in hormone regulation of adenylylcyclase activity.

Human HEK 293 cells present a simple and tractable system to directly test the hypothesis that the G protein gamma subunits contribute to the specificity of receptor signaling pathways in vivo. To begin to elucidate the functions of the individual gamma subunits in these cells, a ribozyme strategy was used to specifically inactivate the mRNA encoding the gamma7 subunit. A phosphorothioated DNA-RNA chimeric hammerhead ribozyme was constructed and analyzed for specificity toward the targeted gamma7 subunit. In vitro cleavage analysis of this ribozyme revealed a highly efficient cleavage activity directed exclusively toward the gamma7 RNA transcript. In particular, this ribozyme did not result in cleavage of the gamma12 RNA transcript, which is 75% identical to the gamma7 RNA transcript. Using a transient transfection assay, in vivo analysis of this ribozyme showed a specific reduction in both the mRNA and protein expression of the gamma7 subunit in HEK 293 cells. Coincident with this loss in gamma7 subunit, there was a specific reduction in the protein expression of the beta1 subunit, suggesting that the beta1 and gamma7 subunits may functionally interact to form a betagamma dimer in vivo. Functional analysis of the consequences of ribozyme-mediated suppression of the gamma7 subunit expression indicated that it was associated with significant attenuation of isoproterenol-, but not prostaglandin E1-, stimulated adenylylcyclase activity. Suppression of the gamma7 subunit expression had no effect on carbachol- and ATP-mediated stimulation of phosphatidylinositol turnover. Taken together, these results not only indicate the feasibility of using the ribozyme technology to determine the roles of individual gamma subunits in receptor-G protein-effector pathways in vivo, but they point to a specific role of the gamma7 subunit in the regulation of adenylylcyclase activity in response to isoproterenol.

G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 1. Reduced membrane-associated G-protein content due to diminished isoprenylation of G-gamma subunits and p21ras.

Mechanisms contributing to altered heterotrimeric G-protein expression and subsequent signaling events during cholesterol accretion have been unexplored. The influence of cholesterol enrichment on G-protein expression was examined in cultured smooth muscle cells that resemble human atherosclerotic cells by exposure to cationized LDL (cLDL). cLDL, which increases cellular free and esterified cholesterol 2-fold and 10-fold, respectively, reduced the cell membrane content of Galphai-1, Galphai-2, Galphai-3, Gq/11, and Galphas. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the Ggamma-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of Galphai subunits was found to be post-transcriptional, since the mRNA steady-state levels of Galphai(1-3) were unchanged following cholesterol enrichment. Second, the membrane expression of alpha and beta subunits was mimicked by cholesterol and 17-ketocholesterol, both of which inhibit HMG-CoA reductase. Third, inhibition of Galphai and Gbeta expression in cholesterol-enriched cells was overcome by mevalonate, the immediate product of HMG-CoA reductase. Fourth, pulse-chase experiments revealed that cholesterol enrichment did not reduce the degradation rate of membrane-associated Galphai subunits. Fifth, cholesterol enrichment also reduced membrane expression of Ggamma-5, Ggamma-7upper; these gamma subunits are responsible for trafficking of the heterotrimeric G-protein complex to the cell membrane as a result of HMG-CoA reductase-dependent post-translational lipid modification (geranylgeranylation) and subsequent membrane association. Cholesterol enrichment did not alter expression of G-gamma-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect in Ggamma subunit expression. Fifth, cholesterol enrichment also reduced the membrane content of p21ras (a low molecular weight G-protein requiring farnesylation for membrane targeting) but did not alter the membrane content of the two proteins that do not require isoprenylation for membrane association&sbd;PDGF-receptor or p60-src. Reduced G-protein content in cholesterol-laden cells was reflected by reduced G-protein-mediated signaling events, including ATP-induced GTPase activity, thrombin-induced inhibition of cyclic AMP accumulation, and MAP kinase activity. Collectively, these results demonstrate that cholesterol enrichment reduces G-protein expression and signaling by inhibiting isoprenylation and subsequent membrane targeting. These results provide a molecular basis for altered G-protein-mediated cell signaling processes in cholesterol-enriched cells.

Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases.

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.

Differential coupling of alpha1-adrenoreceptor subtypes to phospholipase C and mitogen activated protein kinase in neonatal rat cardiac myocytes.

Activation of cardiac alpha1-adrenoreceptors has a number of physiological effects. Ascribing these effects to a specific alpha1-adrenoreceptor subtype first requires the elucidation of the subtypes that are present in the tissue of interest. In the present study, mRNA transcripts for the alpha1A, alpha1B and alpha1D-adrenoreceptor subtypes were detected in cultured neonatal rat cardiac myocytes, using reverse transcriptase-polymerase chain reaction analysis. However, binding sites for only the alpha1A and alpha1B-adrenoreceptor subtypes were detected in cultured neonatal rat cardiac myocytes, using competition binding analysis with a variety of alpha1 selective receptor antagonists. Phenylephrine-stimulated phosphatidylinositol hydrolysis was inhibited by alpha1 selective receptor antagonists with affinities consistent with the alpha1A-adrenoreceptor subtype, whereas phenylephrine-induced activation of the mitogen activated protein kinase cascade was inhibited by these same antagonists with affinities more closely resembling the alpha1B-adrenoreceptor subtype. In the case of both signaling pathways, the alpha1D selective receptor antagonist, BMY 7378, exhibited affinities suggestive of the relative absence of a alpha1D-adrenoreceptor subtype. Thus, despite the presence of mRNA transcripts for all three alpha1-adrenoreceptor subtypes, only the alpha1A and alpha1B-adrenoreceptor subtypes were expressed and functionally coupled at detectable levels in neonatal rat cardiac myocytes. Of particular interest, phenylephrine-induced activation of the mitogen activated protein kinase cascade appears to be mediated by a subtype resembling most closely the pharmacological profile of the alpha1B-adrenoreceptor subtype.

G-protein alpha subunit Gi(alpha)2 mediates erythropoietin signal transduction in human erythroid precursors.

Erythropoietin induces a dose-dependent increase in cytosolic calcium in human erythroblasts that is mediated by a voltage-independent Ca2+ channel. Inhibition of this response to erythropoietin by pertussis toxin suggests involvement of guanine nucleotide-binding regulatory proteins (G-proteins). The role of G-proteins in regulation of the erythropoietin-modulated Ca2+ channel was delineated here by microinjection of G-protein modulators or subunits into human erythroid precursors. This is the first report on the use of microinjection to study erythropoietin signal transduction in normal precursor cells. Fura-2 loaded day-10 burst-forming units-erythroid-derived erythroblasts were used for microinjection and free intracellular calcium concentration ([Ca(i)]) was measured with digital video imaging. BCECF (1,2',7'-bis(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein) was included in microinjectate, and an increase in BCECF fluorescence was evidence of successful microinjection. Cells were microinjected with nonhydrolyzable analogues of GTP, GTPgammaS or GDPbetaS, which maintain the alpha subunit in an activated or inactivated state, respectively. [Ca(i)] increased significantly in a dose-dependent manner after microinjection of GTPgammaS. However, injection of GDPbetaS blocked the erythropoietin-induced calcium increase, providing direct evidence that activation of a G-protein is required. To delineate which G-protein subunits are involved, alpha or betagamma transducin subunits were purified and microinjected as a sink for betagamma or alpha subunits in the erythroblast, respectively. Transducin betagamma, but not alpha, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the alpha subunit. Microinjected antibodies to Gi(alpha)2, but not Gi(alpha)1 or Gi(alpha)3, blocked the erythropoietin-stimulated [Ca(i)] rise, identifying Gi(alpha)2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Gi(alpha)2, but not Gi(alpha)1 or Gi(alpha)3 subunits, to reconstitute the response of pertussis toxin-treated erythroblasts to erythropoietin. These data directly demonstrate a physiologic function of G-proteins in hematopoietic cells and show that Gi(alpha)2 is required in erythropoietin modulation of [Ca(i)] via influx through calcium channels.

Gßγ-Mediated signaling in the heart: Implications of ß and γ subunit heterogeneity.

A family of G proteins, composed of α, ß, and γ subunits, plays a central role in coupling receptors to a variety of enzymes and ion channels. In the cardiovascular system, G proteins are involved in coupling receptors for epinephrine, norepinephrine, acetylcholine, adenosine, angiotensin II, and endothelin to regulation of adenylyl cyclases, phospholipases, and ion channels. For many years, the classic view has been that G protein α subunits provide the requisite specificity for receptor and effector interactions. Recent advances, however, have revealed that the ß and γ subunits also play prominent roles in transducing information from receptors to the appropriate effectors. With the identification of multiple subtypes of ß and γ subunits in the heart, questions are raised regarding their respective roles in signal transduction processes regulating cardiac function.

Isolation of cDNA clones encoding eight different human G protein gamma subunits, including three novel forms designated the gamma 4, gamma 10, and gamma 11 subunits.

With the growing awareness that the G protein beta and gamma subunits directly regulate the activities of various enzymes and ion channels, the importance of identifying and characterizing these subunits is underscored. In this paper, we report the isolation of cDNA clones encoding eight different human gamma subunits, including three novel forms designated gamma 4, gamma 10, and gamma 11. The predicted protein sequence of gamma 4 shares the most identity (60-77%) with gamma 2, gamma 3, and gamma 7 and the least identity (38%) with gamma 1. The gamma 4 is modified by a geranylgeranyl group and is capable of interacting with both beta 1 and beta 2 but not with beta 3. The predicted protein sequence of gamma 10 shows only modest to low identity (35-53%) with the other known gamma subunits, with most of the differences concentrated in the N-terminal region, suggesting gamma 10 may interact with a unique subclass of alpha. The gamma 10 is modified by a geranylgeranyl group and is capable of interacting with beta 1 and beta 2 but not with beta 3. Finally, the predicted protein sequence of gamma 11 shows the most identity to gamma 1 (76% identity) and the least identity to the other known gamma (33-44%). Unlike most of the other known gamma subunits, gamma 11 is modified by a farnesyl group and is not capable of interacting with beta 2. The close resemblance of gamma 11 to gamma 1 raises intriguing questions regarding its function since the mRNA for gamma 11 is abundantly expressed in all tissues tested except for brain, whereas the mRNA for gamma 1 is expressed only in the retina where the protein functions in phototransduction.

Regions outside of the CAAX motif influence the specificity of prenylation of G protein gamma subunits.

A family of GTP-binding regulatory proteins (G proteins) transduces signals across the plasma membrane from a large number of receptors to a smaller number of effectors. Recent studies indicate that a series of post-translational modifications are required for their association with the plasma membrane and for their function. In the case of the G protein gamma subunits, the post-translational modifications include the prenylation of a cysteine residue within a carboxyl-terminal CAAX motif. Although prenylation has been shown to involve the addition of either a C15 farnesyl or a C20 geranylgeranyl group to proteins, the structural requirements and functional consequences of adding different types of prenyl groups to various members of the gamma subunit family have not been examined. In the present study, we have employed the baculovirus expression system to study the structural requirements for attaching different types of prenyl groups to various members of the gamma subunit family. We show that the gamma 2 subunit is modified by a C20 geranylgeranyl group, consistent with the presence of a geranylgeranylation target sequence in this protein. However, we found that the gamma 1 and mutant gamma 2(Ser-71) subunits are modified by both C15 farnesyl and C20 geranylgeranyl groups, despite the presence of an accepted farnesylation target sequence in both of these proteins. Using chimeras of the gamma 1 and gamma 2 subunits, we provide evidence indicating that structural elements upstream of the carboxyl-terminal CAAX motif play a role in the recognition of members of the gamma subunit family by the appropriate insect and mammalian prenyltransferases.

Bovine brain GO isoforms have distinct gamma subunit compositions.

The gamma subunit composition of the major bovine brain Go and Gi proteins (GOA, GOB, GOC, Gi1, and Gi2) was characterized using antibodies against specific gamma isoforms. Each of the purified G protein heterotrimers contained a heterogeneous population of gamma subunits, and the profiles of the gamma subunits found with Gi1, Gi2, and GOA were similar. In contrast, each GO isoform had a distinct pattern of associated gamma subunits. These differences were surprising given that all three alpha O isoforms are thought to share a common amino-terminal sequence important for the binding of beta gamma dimers and that the alpha OA and alpha OC proteins may come from the same alpha O1 mRNA. The free alpha OA and alpha OC subunits had unique elution behaviors during MonoQ chromatography, compatible with differences in their post-translational processing. These results indicate that both the alpha and gamma subunit compositions of heterotrimers define the structure of an intact G protein. Furthermore, the exact subunit composition of G protein heterotrimers may depend upon regulated expression of different subunit isoforms or upon cellular processing of alpha subunits.

Potentiation of Gi-mediated phospholipase C activation by retinoic acid in HL-60 cells. Possible role of G gamma 2.

Differentiated HL-60 cells acquire responsiveness to fMet-Leu-Phe (fMLP), which activates phospholipase C and O2- generation in a pertussis toxin-sensitive manner. Addition of retinoic acid (RA) for the last 24 h during dimethyl sulfoxide (Me2SO)-induced differentiation enhanced fMLP-dependent signals and interaction between fMLP receptor and G(i). RA modifies both the function and subunit composition of G(i)2, the predominant G(i) of HL-60 membranes, as shown by comparing purified G(i)2 from membranes of Me2SO-treated cells (D-G(i)2) to G(i)2 from membranes of cells treated with both Me2SO and RA (DR-G(i)2). As compared to D-G(i)2, DR-G(i)2 induced more fMLP binding when added to membranes of pertussis toxin-treated HL-60 cells and, in the presence of GTP gamma S, stimulated beta gamma-sensitive phospholipase C in extracts of HL-60 cells to a much greater extent at a lower concentrations. Immunoblasts revealed that RA induced expression of the gamma 2 subunit, which was otherwise undetectable in G(i)2 purified from HL-60 cells or in HL-60 membranes. Possibly by inducing expression of gamma 2, RA alters two functions of the G(i) beta gamma subunit, modulation of fMLP receptor-G(i)2 coupling and activation of the effector, Phospholipase C.