Substitution of three amino acids switches receptor specificity of Gqα to that of Giα

Abstract

Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement of GDP bound to the alpha-subunit by GTP. mutations in the C terminus of the alpha-subunit, its covalent modification by pertussis toxin-catalysed ribosylation of ADP, peptide-specific antibodies directed against it, and peptides mimicking C-terminal sequences, all inhibit receptor-mediated activation of G proteins. The logical prediction--that specific amino-acid residues at the C-termini of alpha-subunits can determine the abilities of individual G proteins to discriminate among specific subsets of receptors--has so far not been tested experimentally. Different hormone receptors specifically activate Gq or Gi, whose alpha-subunits (alpha q or alpha i) stimulate phosphatidylinositol-specific phospholipase C or inhibit adenylyl cyclase, respectively. Here we replace C-terminal amino acids of alpha q with the corresponding residues of alpha i2 to create alpha q/alpha i2 chimaeras that can mediate stimulation of phospholipase C by receptors otherwise coupled exclusively to Gi. A minimum of three alpha i2 amino acids, including a glycine three residues from the C terminus, suffices to switch the receptor specificity of the alpha q/alpha i2 chimaeras. We propose that a C-terminal turn, centered on this glycine, plays an important part in specifying receptor interactions of G proteins in the Gi/Go/Gz family.

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

“…Both plasmids contained a short sequence coding for an internal hemagglutinin (HA) epitope tag (DVPDYA), which replaced WTq residues 125-130 (18). The presence of the epitope tag did not affect the receptor and effector coupling properties of WTq (8,9,18). The identity of the two G protein constructs was verified by dideoxy sequencing (19).…”

“…Protein concentrations were determined using the Bio-Rad protein assay kit with IgG as the standard. 8 ]vasopressin, (Ϫ)-isoproterenol, and PTX were purchased from Sigma. All other ligands used in this study were obtained through Research Biochemicals Inc.…”

Surface expression of metabotropic glutamate receptor variants mGluR1a and mGluR1b in transfected HEK293 cells

“…To allow detection of the recombinant receptors on cell surface the N-terminal 16 residues of rat GB1a and N-terminal 41 residues of rat GB2 were replaced by 36 residues encoding the mGluR5 signal peptide, and HA epitope. The adaptor Gqi9 protein, based on sequence of Gq with last 9 residues replaced by extreme C terminal 9 residues from Gi sequence was described (Conklin et al, 1993) and used previously in our studies (Franek et al, 1999;Galvez et al, 2001;Havlickova et al, 2003). All constructs were sequenced using Big Dye Terminater v. 3,1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).…”

μ-Opioid Receptor Forms a Functional Heterodimer With Cannabinoid CB1 Receptor: Electrophysiological and FRET Assay Analysis

“…The chimeric G qi5 protein, whose last five amino acid residues in the C-terminus was replaced with the corresponding portion of G i protein, is useful for monitoring the responses mediated by stimulation of G i / o protein-coupled receptors in the Xenopus oocyte expression system, as reported previously by our laboratory (10,11). Chimeric G qi5 allows G i / o -coupled receptors to couple to the phospholipase C (PLC)-mediated signal pathway (12,13). Agonists for G i / o protein-coupled receptors can elicit Ca 2+ -activated Cl − currents in such oocytes only through the chimeric G qi5 , but not through G i / o endogenously expressed in oocytes (10,11).…”