Efficient Interaction with a Receptor Requires a Specific Type of Prenyl Group on the G Protein γ Subunit

Kisselev, Oleg G.; Ермолаева, М В; Gautam, N.

doi:10.1074/jbc.270.43.25356

Cited by 144 publications

(149 citation statements)

References 24 publications

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“…Mutations in the homologous γ1 subunit C terminal domain prevented Gt, the G protein in rod photoreceptors, from binding to activated rhodopsin [14]. A peptide specific to the C terminal domain of γ1 stabilized the activated form of rhodopsin but when the amino acids in the peptide were scrambled it was inactive [33]. Evidence from other experiments also supports a role for this C terminal γ subunit domain with a receptor [7,34].…”

Section: The γ Subunit C Terminus Is Likely Required For Retention Ofmentioning

confidence: 84%

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

Akgöz¹,

Kalyanaraman²,

Gautam³

2006

Cellular Signalling

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On activation of a receptor the G protein βγ complex translocates away from the receptor on the plasma membrane to the Golgi complex. The rate of translocation is influenced by the type of γ subunit associated with the G protein. Complementary approaches -imaging living cells expressing fluorescent protein tagged G proteins and assaying reconstituted receptors and G proteins in vitrowere used to identify mechanisms at the basis of the translocation process. Translocation of Gβγ containing mutant γ subunits with altered prenyl moieties showed that the differences in the prenyl moieties were not sufficient to explain the differential effects of geranylgeranylated γ5 and farnesylated γ11 on the translocation process. The translocation properties of Gβγ were altered dramatically by mutating the C terminal tail region of the γ subunit. The translocation characteristics of these mutants suggest that after receptor activation, Gβγ retains contact with a receptor through the γ subunit C terminal domain and that differential interaction of the activated receptor with this domain controls Gβγ translocation from the plasma membrane.

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Section: The γ Subunit C Terminus Is Likely Required For Retention Ofmentioning

confidence: 84%

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

Akgöz¹,

Kalyanaraman²,

Gautam³

2006

Cellular Signalling

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“…Multiple studies indicate that the composition of the prenyl group on the C terminus of the ␥ subunit is very important for the interaction of the ␤␥ dimer with receptors and effectors (34,(55)(56)(57)(58). For this reason we tested the role of the prenyl group in the activation of PtdIns 3-kinase by using ␥ subunits that contained altered CAAX boxes to direct the 50 …”

Section: Fig 1 Purification Of Ptdins 3-kinase and G␤␥ From Sf9 Cellsmentioning

confidence: 99%

Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

Kerchner

Clay

McCleery

et al. 2004

Journal of Biological Chemistry

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The ability of G protein ␣ and ␤␥ subunits to activate the p110␥ isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110␥ form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTPactivated G s , G i , G q , or G o ␣ subunits were unable to activate PtdIns 3-kinase. Dimers containing G␤ 1-4 complexed with ␥ 2 -stimulated PtdIns 3-kinase activity about 26-fold with EC 50 values ranging from 4 to 7 nM. G␤ 5 ␥ 2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase C␤. A series of dimers with ␤ subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of ␤␥ with phosducin (␤1H311A␥2, ␤1R314A␥2, and ␤1W332A␥2) was tested, and only ␤1W332A␥2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the ␤ 1 subunit complexed with a panel of different G␥ subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The ␤ 1 ␥ 2 , ␤ 1 ␥ 10 , ␤ 1 ␥ 12 , and ␤ 1 ␥ 13 dimers all activated PtdIns 3-kinase about 26-fold with 4 -25 nM EC 50 values. The ␤ 1 ␥ 11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110␥ form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the ␥ subunit in determining the activation of PtdIns 3-kinase was examined using ␥ subunits with altered CAAX boxes directing the addition of farnesyl to the ␥ 2 subunit and geranylgeranyl to the ␥ 1 and ␥ 11 subunits. Replacement of the geranylgeranyl group of the ␥ 2 subunit with farnesyl inhibited the activity of ␤ 1 ␥ 2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the ␥ 1 and ␥ 11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all ␤ subunits, with the exception of ␤ 5 , interact equally well with PtdIns 3-kinase. In contrast, the composition of the ␥ subunit and its prenyl group markedly affects the ability of the ␤␥ dimer to stimulate PtdIns 3-kinase.The generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) 1 in the inner leaflet of the plasma membrane is critical to the regulation of cell function (1-3). The phosphorylated inositol head group provides a docking site for proteins containing pleckstrin homology domains (PH domains) and leads to activation of many enzymes including the phosphoinositol-dependent protein kinase and protein kinase B (Akt). Activation of protein kinase B regulates multiple cellular functions including differentiation, regulation of metabolic events, cell survival, and motility (1-3). In keeping with this central role, the level of PIP3 is tightly regulated, it can be elevated by multiple classes of receptors (1, 2), and there are specific phosphatidylinositol 5-phosphatases (SHIP and PTEN) that degrade the signal (4 -8).A large family of phosphatidylinositol 4,5-bisphosphate 3-kinases (PtdIns 3-kinases) is responsible for generating PIP3 by phosphorylating the D3 ...

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“…Given the nature of the physical linkage between the receptor and G protein in the fusion proteins, the knowledge that the N terminus of the G protein ␣ subunit plays a central role in interaction with the ␤␥ complex (28 -29), the concept that the ␤␥ complex may play a key role in receptor interactions with the ␣ subunit (33)(34)(35), and the understanding that G protein ␣ subunit acylation is important in interactions with the ␤␥ complex, it was of considerable interest to observe that coexpression of excess ␤ 1 ␥ 2 along with any of the ␣ 2A -adrenoreceptor-G i1 ␣ fusion proteins resulted in greater maximal UK14304 stimulation of GTPase activity (Fig. 5).…”

Section: Fig 4 Uk14304 Stimulates High Affinity Gtpase Activity Of mentioning

confidence: 99%

Rescue of Functional Interactions between the α2A-Adrenoreceptor and Acylation-resistant Forms of Gi1α by Expressing the Proteins from Chimeric Open Reading Frames

Wise¹,

Milligan

1997

Journal of Biological Chemistry

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Co-expression of the ␣ 2A -adrenoreceptor with a pertussis toxin-resistant (C351G), but not with an also palmitoylation-resistant (C3S/C351G), form of the ␣ subunit of G i1 resulted in agonist-induced, pertussis toxinindependent, GTP hydrolysis. Construction and expression of a chimeric fusion protein between the receptor and C351G G i1 ␣ generated a membrane protein in which the G protein element was activated by receptor agonist. An equivalent fusion protein containing C3S/C351G G i1 ␣ rescued the ability of receptor agonist to activate this mutant. Fusion proteins of a palmitoylation-resistant (C442A) ␣ 2A -adrenoreceptor and either C351G or C3S/ C351G G i1 ␣ also responded effectively to agonist. Myristoylation resistant (G2A/C351G) and combined acylation-resistant (G2A/C3S/C351G) mutants of G i1 ␣ are cytosolic proteins. Expression of these as chimeric ␣ 2A -adrenoreceptor-G protein fusions restored membrane localization and activation of the G protein by receptor agonist. These studies demonstrate the general utility of generating chimeric fusion proteins to examine receptor regulation of G protein function and that the lack of functional activation of acylation-negative G proteins by a co-expressed receptor is related to deficiencies in cellular targeting and location rather than an inherent incapacity to produce appropriate protein-protein interactions and signal transmission.A major mechanism for signal transduction across the plasma membrane involves seven transmembrane element G protein-coupled receptors (GPCRs) 1 and their activation of members of the family of ␣␤␥ heterotrimeric guanine nucleotide binding proteins (G proteins) (1-2). Unlike the GPCRs, none of the individual G protein subunits contain transmembrane-spanning elements although the proteins are membrane-associated. In the case of the ␤␥ complex, post-translational prenylation by the C15 farnesyl or C20 geranylgeranyl groups at a cysteine residue close to the C-terminal tail of the ␥ subunit followed by protein trimming and carboxymethylation acts to anchor the complex to the membrane (3-4).A key role in the targeting of specific G protein ␣ subunits to the plasma membrane and their maintenance there has been ascribed to both co-translational myristoylation and posttranslational palmitoylation (3-13). These acylations may also contribute to protein-protein interactions between the G protein ␣ subunit and both receptors and the G protein ␤␥ complex (12,14). Addition of myristate occurs only on the ␣ subunits of the G i -family of G proteins because they contain the consensus sequence (MGXXXS) for the enzyme N-myristoyl-CoA transferase. The glycine that is found at codon 2 acts as the acceptor when it is exposed following removal of the initiator methionine. Palmitoylation of either one or two cysteine residues within the first ten amino acids of the ␣ subunits occurs on all of the widely expressed G proteins (4, 11).Although receptors can often be resolved from their cognate G proteins by detergent solubilization of membranes, in a number...

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Efficient Interaction with a Receptor Requires a Specific Type of Prenyl Group on the G Protein γ Subunit

Cited by 144 publications

References 24 publications

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

Rescue of Functional Interactions between the α2A-Adrenoreceptor and Acylation-resistant Forms of Gi1α by Expressing the Proteins from Chimeric Open Reading Frames

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