<i>In vitro</i> mutagenesis and the search for structure-function relationships among G protein-coupled receptors

Savarese, Todd M.; Fraser, Claire M.

doi:10.1042/bj2830001

Cited by 462 publications

(255 citation statements)

References 166 publications

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“…Our observation that three different competing peptides affect AT "A R-G-protein coupling is consistent with the hypothesis that multiple regions of the receptor form a three-dimensional binding surface to which the G-protein is coupled [8,14,28]. In the AT "A R, this binding surface comprises at least regions from i2, i3 and the C-terminal tail.…”

Section: Discussionsupporting

confidence: 89%

“…Palmitoylation of cysteine-322 in the C-terminal tail of the β-adrenergic receptors is speculated to form a short intracellular loop by embedding the palmitoyl moiety in the membrane. Since the amphiphilic helix is in this small loop and since loop formation in the proximal C-terminal tail is also found in many G-protein-coupled receptors, it was suggested that the small loop structure in the C-terminal tail may be significant in the receptor-G-protein interaction [28]. In the AT "A R, a consensus palmitoylation site occurs in the distal region of the C-terminal tail [29], suggesting that, for this receptor, a stimulated loop structure would be quite large.…”

Section: Discussionmentioning

confidence: 99%

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G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence

Huang

Ushio‐Fukai

et al. 1998

Biochemical Journal

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The vascular angiotensin II type 1 receptor (AT1AR) is a member of the G-protein-coupled receptor superfamily. We mapped the G-protein binding domains of the AT1AR using synthetic peptides selected from the receptor sequence, which interfere with AT1AR–G-protein coupling. Membrane GTPase activity was used as a measure of the functional coupling in rat vascular smooth muscle cells. Peptides corresponding to the N-terminal region of the second intracellular loop (residues 125–137), the N-terminal region of the third intracellular loop (217–227) and the juxtamembranous region of the C-terminal tail (304–316) inhibited angiotensin II-induced GTPase activation by 30%, 30%, and 70%, respectively. The latter two domains (217–227 and 304–316) are predicted to form amphiphilic α-helices. Only the peptide representing residues 217–227 stimulated basal activity (45%). No synthetic peptide had a significant effect on either the number or the affinity of the AT1AR binding. These observations indicate that domains of the second and third regions and the cytoplasmic tail of the AT1AR interact with G-proteins, and that multiple contacts with these receptor domains may be important for binding and activation of the G-proteins.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence

Huang

Ushio‐Fukai

et al. 1998

Biochemical Journal

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“…Therefore, *Corresponding author. Fax: (49) (6421) 28-8924. speculations about GIP receptor defects arose which find some support by a number of human diseases that can be attributed to point mutations in G protein-linked receptors [4,5,6]. Such defects alter either agonist binding, G protein-receptor interactions, or cause inproper membrane incorporation of the receptor [5,7].…”

Section: Introductionmentioning

confidence: 99%

Molecular cloning, functional expression, and signal transduction of the GIP‐receptor cloned from a human insulinoma

et al. 1995

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“…G protein-coupled receptors (GPCRs), 1 when activated by extracellular ligands, interact with specific classes of heterotrimeric G proteins (consisting of ␣, ␤, and ␥ subunits) which can then, in their activated forms, inhibit or activate various effector enzymes and/or ion channels (1)(2)(3)(4)(5). Characteristically, a specific GPCR can interact with only a limited subset of the many structurally similar G proteins that are expressed within a cell.…”

mentioning

confidence: 99%

The N-terminal Extension of Gαq Is Critical for Constraining the Selectivity of Receptor Coupling

Kostenis

Degtyarev²,

Conklin³

et al. 1997

Journal of Biological Chemistry

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Characteristically, an individual member of the superfamily of G protein-coupled receptors can interact only with a limited number of the many structurally closely related G protein heterotrimers that are expressed within a cell. Interestingly, the N termini of two G protein ␣ subunits, G␣ q and G␣ 11 , differ from those of other ␣ subunits in that they display a unique, highly conserved six-amino acid extension. To test the hypothesis that this sequence element is critical for proper receptor recognition, we prepared a G␣ q deletion mutant (؊6q) lacking these first six amino acids. The ؊6q construct (or wild type G␣ q as a control) was coexpressed (in COS-7 cells) with several different G i/o -or G s -coupled receptors, and ligand-induced increases in inositol phosphate production were determined as a measure of G protein activation. Whereas these receptors did not efficiently interact with wild type G␣ q , most of them gained the ability to productively couple to ؊6q. Additional experiments indicated that the observed functional promiscuity of ؊6q is not due to overexpression (as compared with wild type G␣ q ) or to a lack of palmitoylation. We conclude that the N-terminal extension characteristic for G␣ q/11 proteins is critical for constraining the receptor coupling selectivity of these subunits, indicative of a novel mechanism by which the fidelity of receptor-G protein interactions can be regulated.G protein-coupled receptors (GPCRs), 1 when activated by extracellular ligands, interact with specific classes of heterotrimeric G proteins (consisting of ␣, ␤, and ␥ subunits) which can then, in their activated forms, inhibit or activate various effector enzymes and/or ion channels (1-5). Characteristically, a specific GPCR can interact with only a limited subset of the many structurally similar G proteins that are expressed within a cell. Molecular genetic and biochemical studies have identified distinct intracellular regions (as well as single amino acids contained within these domains) on the GPCR proteins that play key roles in determining the fidelity of receptor-G protein coupling (1-7). In addition, recent studies have shown that residues at the extreme C terminus of the G protein ␣ subunits are also of fundamental importance for the selectivity of receptor-G protein interactions (8 -10). However, several lines of evidence suggest that the C terminus of the G␣ subunits is clearly not the only structural determinant on the G proteins that is critical for dictating receptor-G protein coupling selectivity (2, 5).Interestingly, two G protein ␣ subunits, G␣ q and G␣ 11 , contain a unique six-amino acid extension that is not found in other G␣ subunits (Fig. 1). This short sequence is highly conserved among all vertebrate species from which these subunits have been cloned so far (11)(12)(13)(14)(15), suggesting that it may be relevant for some aspect of G␣ q /G␣ 11 function.Previous studies (16,17) analyzing the biochemical properties of a mutant G␣ q subunit lacking the N-terminal extension (hereafter referred...

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In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors

Cited by 462 publications

References 166 publications

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence

Molecular cloning, functional expression, and signal transduction of the GIP‐receptor cloned from a human insulinoma

The N-terminal Extension of Gαq Is Critical for Constraining the Selectivity of Receptor Coupling

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