Primary Structure of a γ Subunit of G Protein, γ12, and Its Phosphorylation by Protein Kinase C

Morishita, Rika; Nakayama, Hiroshi; Isobe, Toshiaki; Matsuda, Takehisa; Hashimoto, Yuichi; Okano, Toshiyuki; Fukada, Yoshitaka; Mizuno, Keiko; Ohno, Shigeo; Kozawa, Osamu; Kato, Kanefusa; Asano, Taku

doi:10.1074/jbc.270.49.29469

Cited by 83 publications

(78 citation statements)

References 50 publications

(57 reference statements)

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“…These observations are compatible with recent studies on immunological identification of both ␣ and ␤ subunits of trimeric G-proteins in the secretory granule fraction of normal rat islets and insulinoma tissue (22), and ␤TC3 cells (48), and provide further evidence for not only the presence of all three subunits of ␣␤␥ trimer on secretory granules, but also their (functional) regulation by carboxyl methylation (this study) and phosphorylation (32). Immunoblotting data indicated that at least four isoforms of ␥ subunits (i.e., ␥ 1 , ␥ 2 , ␥ 5 , and ␥ 7 ) are localized in pancreatic ␤ cells (Fig.…”

Section: Discussionsupporting

confidence: 80%

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

Kowluru¹,

Li²,

Metz³

1997

J. Clin. Invest.

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The ␥ subunits of trimeric G-proteins ( ␥ 1 , ␥ 2 , ␥ 5 , and ␥ 7 isoforms) were found to be methylated at their carboxyl termini in normal rat islets, human islets and pure ␤ [HIT-T15] cells. Of these, GTP ␥ S significantly stimulated the carboxyl methylation selectively of ␥ 2 and ␥ 5 isoforms. Exposure of intact HIT cells to either of two receptor-independent agonists-a stimulatory concentration of glucose or a depolarizing concentration of K ϩ -resulted in a rapid (within 30 s) and sustained (at least up to 60 min) stimulation of ␥ subunit carboxyl methylation. Mastoparan, which directly activates G-proteins (and insulin secretion from ␤ cells), also stimulated the carboxyl methylation of ␥ subunits in intact HIT cells. Stimulatory effects of glucose or K ϩ were not demonstrable after removal of extracellular Ca 2 ϩ or depletion of intracellular GTP, implying regulatory roles for calcium fluxes and GTP; however, the methyl transferase itself was not directly activated by either. The stimulatory effects of mastoparan were resistant to removal of extracellular Ca 2 ϩ , implying a mechanism of action that is different from glucose or K ϩ but also suggesting that dissociation of the ␣␤␥ trimer is conducive to ␥ subunit carboxyl methylation. Indeed, pertussis toxin also markedly attenuated the stimulatory effects of glucose, K ϩ or mastoparan without altering the rise in intracellular calcium induced by glucose or K ϩ . Glucose-induced carboxyl methylation of ␥ 2 and ␥

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Section: Discussionsupporting

confidence: 80%

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

Kowluru¹,

Li²,

Metz³

1997

J. Clin. Invest.

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“…S. Mumby and A. G. Gilman (23). The antibodies against various ␥ subunits have been described previously (11,24). The isoform specificity and immunoprecipitation efficiency of antibodies used in this study are shown in Table I.…”

Section: Methodsmentioning

confidence: 99%

“…The ␤␥ complex was also found to be involved in the stimulation of mitogen-activated protein kinase (3,4). So far, seven ␤ (from five separate genes and including two splice variants, ␤ 5L and ␤ 3S ) and 11 ␥ subunit isoforms have been found (1,(5)(6)(7)(8)(9)(10)(11). Different ␤␥ complexes do not have equivalent abilities to couple ␣ subunits to receptors or to regulate effectors.…”

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confidence: 99%

Selective Association of G Protein β4 with γ5 and γ12 Subunits in Bovine Tissues

Asano

Morishita

Ueda

et al. 1999

Journal of Biological Chemistry

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The ␤ and ␥ subunits of G proteins are tightly bound under physiological conditions, and so far, seven ␤ and 11 ␥ subunit isoforms have been found. The relative abilities of the ␤ and ␥ subunits to associate with each other have been studied using transfected cell assays, in vitro translation and the yeast two-hybrid system, but have not been fully characterized in various tissues. In the present study, we demonstrated the selectivity of association of the ␤ with ␥ isoforms in bovine tissues. Immunoprecipitation of ␤␥ complexes from tissue extracts with antibodies against various ␥ subunits and subsequent analyses revealed that ␤ 4 associated with the ␥ subunits with the following rank order of selectivity: ␥ 5 > ␥ 12 > ␥ 2 > ␥ 3 , while ␤ 2 bound to ␥ 2 , ␥ 3 , and ␥ 12 more selectively than to ␥ 5 . By contrast, ␤ 1 associated with all ␥ subunits without significant selectivity. Analyses of purified ␤␥ complexes containing various ␥ isoforms revealed ␤ subunit compositions similar to those found in the immunoprecipitates. Particular combinations of ␤ and ␥ subunit isoforms may contribute to maintaining efficient and specific signal transduction mediated by G proteins. Heterotrimeric G proteins1 are composed of ␣, ␤, and ␥ subunits, the latter two being tightly associated under physiological conditions (1, 2). Receptor stimulation promotes the dissociation of G protein into a GTP-bound ␣ subunit and a ␤␥ dimer, both of which independently regulate intracellular effectors. Many studies have shown that the ␤␥ dimer regulates adenylyl cyclase, phospholipase C␤, phosphatidylinositol 3-kinase ␥, receptor kinase, K ϩ channels, and Ca 2ϩ channels (1, 2). The ␤␥ complex was also found to be involved in the stimulation of mitogen-activated protein kinase (3, 4). So far, seven ␤ (from five separate genes and including two splice variants, ␤ 5L and ␤ 3S ) and 11 ␥ subunit isoforms have been found (1, 5-11). Different ␤␥ complexes do not have equivalent abilities to couple ␣ subunits to receptors or to regulate effectors. For example, the ␤ 1 and ␤ 3 are selectively involved in the signal transduction cascades from somatostatin and muscarinic M4 receptors, respectively, to voltage-dependent Ca 2ϩ channels (12). The ␤␥ complex containing ␥ 1 is less effective than ␤␥ complexes containing the other ␥ subunits in inhibiting calmodulin-stimulated adenylyl cyclase activity, enhancing G␣ sstimulating type II adenylyl cyclase activity, stimulating phospholipase C␤ 3 activity, and stimulating the cardiac K ϩ channel (13-15). These data suggest that cellular responses to receptor stimulation may vary as a function of the ␤␥ complexes found in the target cells.The selectivity of ␤ and ␥ subunit interactions has been determined using transfected cell assay systems (16) and in vitro translation systems (10,17). These studies demonstrated that ␤ 1 binds all ␥ subunits, ␤ 2 binds most ␥ isoforms but not ␥ 1 and ␥ 11 , and ␤ 3 does not bind any ␥ subunits. Recently, the yeast two-hybrid system was applied to obtain a more sensitive measu...

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“…Recently, the ␥ 12 subunit has been shown to be a substrate for protein kinase C (12,13), suggesting that dimers containing this ␥ subunit may be subject to regulation by phosphorylation. The ␥ 12 subunit is widely expressed (12)(13)(14)(15) and, given the extensive role of the ␤␥ subunit in cell signaling (6,16), its phosphorylation may have important consequences. To examine the effects of ␥ 12 subunit phosphorylation on its activity, we purified recombinant ␤ 1 ␥ 12 dimers from baculovirus-infected Sf9 insect cells, phosphorylated them with PKC ␣ and ␤ 1 , and tested their activity in three assays of ␤␥ function.…”

mentioning

confidence: 99%

Phosphorylation of the G Protein γ12 Subunit Regulates Effector Specificity

Yasuda¹,

Lindorfer

Myung

et al. 1998

Journal of Biological Chemistry

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Although the G protein ␤␥ dimer is an important mediator in cell signaling, the mechanisms regulating its activity have not been widely investigated. The ␥ 12 subunit is a known substrate for protein kinase C, suggesting phosphorylation as a potential regulatory mechanism. Therefore, recombinant ␤ 1 ␥ 12 dimers were overexpressed using the baculovirus/Sf9 insect cell system, purified, and phosphorylated stoichiometrically with protein kinase C ␣. Their ability to support coupling of the G i1 ␣ subunit to the A1 adenosine receptor and to activate type II adenylyl cyclase or phospholipase C-␤ was examined. Phosphorylation of the ␤ 1 ␥ 12 dimer increased its potency in the receptor coupling assay from 6.4 to 1 nM, changed the K act for stimulation of type II adenylyl cyclase from 14 to 37 nM, and decreased its maximal efficacy by 50%. In contrast, phosphorylation of the dimer had no effect on its ability to activate phospholipase C-␤. The native ␤ 1 ␥ 10 dimer, which has 4 similar amino acids in the phosphorylation site at the N terminus, was not phosphorylated by protein kinase C ␣. Creation of a phosphorylation site in the N terminus of the protein (Gly 4 3 Lys) resulted in a ␤ 1 ␥ 10G4K dimer which could be phosphorylated. The activities of this ␤␥ dimer were similar to those of the phosphorylated ␤ 1 ␥ 12 dimer. Thus, phosphorylation of the ␤ 1 ␥ 12 dimer on the ␥ subunit with protein kinase C ␣ regulates its activity in an effector-specific fashion. Because the ␥ 12 subunit is widely expressed, phosphorylation may be an important mechanism for integration of the multiple signals generated by receptor activation.Most cells possess multiple signaling pathways to receive signals from the hormones, autacoids, neurotransmitters, and growth factors in their environment. One of the best characterized signal transduction systems is used by receptors coupled to the heterotrimeric G proteins 1 (1-5). Receptors activate this system by stimulating the release of bound GDP from the G protein ␣ subunit leading to exchange of GDP for GTP in the protein's nucleotide binding site. Binding of GTP induces a conformational change in the ␣ subunit, simultaneously activating the protein and markedly decreasing its affinity for the ␤␥ dimer (1). Both the GTP-bound form of the ␣ subunit and the released ␤␥ subunit are capable of activating multiple effectors to generate intracellular messages (3, 4, 6, 7). The mechanisms that regulate the lifetime of the active, GTPbound form of the ␣ subunit have been studied extensively. All ␣ subunits have an intrinsic GTPase activity, which hydrolyzes bound GTP to GDP (1, 3, 4), returning the molecule to its basal state and increasing its affinity for GDP and the ␤␥ subunit (1,3,4,8). Both changes induce formation of the stable, heterotrimeric form of the G protein. Interestingly, the GTPase activity of many ␣ subunits can be increased by a class of proteins termed RGS molecules (9, 10) and by certain effectors such as PLC-␤ (11). Although the activity of the ␣ subunit is regulated by multiple me...

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Primary Structure of a γ Subunit of G Protein, γ12, and Its Phosphorylation by Protein Kinase C

Cited by 83 publications

References 50 publications

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

Selective Association of G Protein β4 with γ5 and γ12 Subunits in Bovine Tissues

Phosphorylation of the G Protein γ12 Subunit Regulates Effector Specificity

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