Abstract

₁-adrenergic receptors (₁-ARs) are members of the G-protein-coupled receptor (GPCR) superfamily and activate inositol phosphate (IP) turnover. We show that glycine and asparagine mutations of Phe303 in transmembrane segment VI (TMVI) of the _1B-AR, a highly conserved residue in GPCRs, although increasing agonist affinity, abolish agonist-activated IP signalling. Co-expression of the Phe303 mutants also inhibited (–)epinephrine-stimulated IP signalling by wild-type _1B-AR and other G_q-coupled receptors, as well as IP signalling mediated by AlF₄^- stimulation of both wild-type G_q and a constitutively active mutant. The inability of the Phe303 mutants to signal is due to induction of a receptor conformation that dissociates G-protein binding from activation. As a result, the Phe303 mutants sequester G_q and stoichiometrically inhibit Gq signalling in a dominant-negative manner. We further show that both the enhanced basal and agonist-stimulated IP-signalling activity of the constitutively active _1B-AR mutants, C128F and A293E, are inhibited in the double mutants, C128F/F303G and A293E/F303G. Phe303, therefore, appears to be critically involved in coupling TMVI -helical movement, a key step in receptor activation, to activation of the cognate G-protein.