ACTIVATION OF G(I) PROTEIN BY PEPTIDE STRUCTURES OF THE MUSCARINIC M(2) RECEPTOR 2ND INTRACELLULAR LOOP

The muscarinic M(2) receptor that normally couples via G(i) to inhibit adenylyl cyclase was made to couple to G(s) by exchange of its third intracellular loop for the comparable domain of the beta(2)-adrenocept or. In HeLa cells transfected with the recombinant M(2) beta i-3 cDNA, the chimaeric receptor showed carbachol-mediated activation of adenyl yl cyclase (EC(50) = 73 nM) that was blocked by atropine, but not by p ropranolol. The chimaeric receptor was shown to mediate a carbachol-st imulated, Bordetella pertussis toxin-sensitive GTPase activity in memb ranes of transfected HeLa cells. Interestingly, stimulation of adenyly l cyclase by carbachol was 2-fold higher in transfected cells that had been pretreated with pertussis toxin. These data suggested that the M (2) beta i-3 receptor was able to couple to both G(i) and G(s), and th at the ability to recognise and stimulate G(i) did not involve the thi rd cytoplasmic loop of M(2). We investigated peptide elements taken fr om the second intracellular loop of the M(2) receptor for their abilit y to mediate activation of G(i) and found that a nine amino acid pepti de representing the C-terminal sequence, VKRTTKMAG-NH2 (V9G), was capa ble of inhibiting forskolin-stimulated adenylyl cyclase by up to 18% a nd could stimulate high affinity GTPase activity of rat brain membrane s by 32%. Further, V9G was shown to cause a doubling of the initial ra te of [S-35]GTP gamma S binding to purified bovine brain G(i)/G(o) in reconstituted phospholipid vesicles. These data identify a domain on t he second intracellular loop of the muscarinic M(2) receptor that is i nvolved in the selection of a pertussis toxin-sensitive G protein.