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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Agonist-stimulated high affinity GTPase activity of fusion proteins between the ␣ 2A -adrenoreceptor and the ␣ subunits of forms of the G proteins G i1 , G i2 , G i3 , and G o1 , modified to render them insensitive to the action of pertussis toxin, was measured following transient expression in COS-7 cells. Addition of a recombinant regulator of G protein signaling protein, RGS4, did not significantly affect basal GTPase activity nor agonist stimulation of the fusion proteins containing G␣ i1 and G␣ i3 but markedly enhanced agonist-stimulation of the proteins containing G␣ i2 and G␣ o1. The effect of RGS4 on the ␣ 2A -adrenoreceptor-G␣ o1 fusion protein was concentration-dependent with EC 50 of 30 ؎ 3 nM and the potency of the receptor agonist UK14304 was reduced 3-fold by 100 nM RGS4. Equivalent reconstitution with Asn 88 -Ser RGS4 failed to enhance agonist function on the ␣ 2A -adrenoreceptor-G␣ o1 or ␣ 2A -adrenoreceptor-G␣ i2 fusion proteins. Enzyme kinetic analysis of the GTPase activity of the ␣ 2A -adrenoreceptor-G␣ o1 and ␣ 2A -adrenoreceptor-G␣ i2 fusion proteins demonstrated that RGS4 both substantially increased GTPase V max and significantly increased K m of the fusion proteins for GTP. The increase in K m for GTP was dependent upon RGS4 amount and is consistent with previously proposed mechanisms of RGS function. Agonist-stimulated GTPase turnover number in the presence of 100 nM RGS4 was substantially higher for ␣ 2A -adrenoreceptor-G␣ o1 than for ␣ 2A -adrenoreceptor-G␣ i2 . These studies demonstrate that although RGS4 has been described as a generic stimulator of the GTPase activity of G i -family G proteins, selectivity of this interaction and quantitative variation in its function can be monitored in the presence of receptor activation of the G proteins.Initiation of signal transduction cascades involving heterotrimeric G proteins requires that the binding of an agonist ligand to a G protein-coupled receptor (GPCR) 1 results in the stabilization of conformations of the GPCR which increase the rate of dissociation of bound GDP from the nucleotide binding pocket of the ␣ subunit of a cognate G protein. Binding of GTP is thus allowed. With GTP bound the G protein produces a series of activating conformational changes and can thence regulate, directly or otherwise, the activity of several enzymes which generate intracellular second messengers or the probability of opening of a range of ion channels (1). Effector regulation is terminated by the intrinsic GTPase activity of the G protein ␣ subunit. Although this cycle of events is clear (1), it is only in recent years that explanations for the measured discrepancy in GTPase activity rates of heterotrimeric G proteins, which appeared too slow to account for biochemically and electrophysiologically measured functional end points, have begun to become apparent. A number of effector enzymes have been shown to display GTPase activating protein (GAP) activity toward their partner G proteins (2-4). Moreover, a relatively recently identified family of regu...

Section: Construct Gtp K M (Nm)mentioning

confidence: 65%

Section: Construct Gtp K M (Nm)mentioning

confidence: 99%

The Regulator of G Protein Signaling RGS4 Selectively Enhances α2A-Adreoreceptor Stimulation of the GTPase Activity of Go1α and Gi2α

Druey²,

Milligan³

2000

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“…These have included linking the G protein to a single transmembrane-spanning element of a GPCR (50). However, the current approach ensures proximity of the G protein to the GPCR and has been successfully applied previously for acylation-resistant forms of G␣ i1 (51).…”

Section: Discussionmentioning

confidence: 99%

Coordinated Agonist Regulation of Receptor and G Protein Palmitoylation and Functional Rescue of Palmitoylation-deficient Mutants of the G Protein G11α following Fusion to the α1b-Adrenoreceptor

Stevens¹,

Pediani

Carrillo

et al. 2001

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Transfection of either the ␣ 1b -adrenoreceptor or G␣ 11 into a fibroblast cell line derived from a G␣ q /G␣ 11 double knockout mouse failed to produce elevation of intracellular [Ca 2؉ ] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the ␣ 1b -adrenoreceptor with the palmitoylation-resistant C9S,C10S G␣ 11 also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S G␣ 11 or C10S G␣ 11 . Expression of a fusion protein between the ␣ 1b -adrenoreceptor and G␣ 11 allowed [Ca 2؉ ] i elevation, and this was also true for a fusion protein between the ␣ 1b -adrenoreceptor and C9S,C10S G␣ 11 , since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin ␣, as a ␤⅐␥-sequestering agent, fully attenuated the Ca 2؉ signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type G␣ 11 were also targets for agonist-regulated

“…Furthermore, GFP is not the only protein that can be attached to the COOH-terminal tail of a G protein-coupled receptor without disrupting function. We and others have produced fusion proteins in which the ␣ subunits of heterotrimeric G proteins have been attached directly to the COOH-terminal tail of various receptors (42)(43)(44)(45)(46). From the long isoform of the rat TRH receptor, we generated for these studies both an NH 2 -terminally epitope-tagged form of the receptor (VSV-TRHR) and subsequently the final chimeric protein (VSV-TRHR-GFP).…”

Section: Discussionmentioning

confidence: 99%

Real Time Visualization of Agonist-mediated Redistribution and Internalization of a Green Fluorescent Protein-tagged Form of the Thyrotropin-releasing Hormone Receptor

Drmota

Gould

Milligan

1998

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