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)-adrenoceptor. In HeLa cells transfected with the recombinant M(2) beta i-3 cDNA, the chimaeric receptor showed carbachol-mediated activation of adenylyl cyclase (EC(50) = 73 nM) that was blocked by atropine, but not by propranolol. The chimaeric receptor was shown to mediate a carbachol-stimulated, Bordetella pertussis toxin-sensitive GTPase activity in membranes of transfected HeLa cells. Interestingly, stimulation of adenylyl 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 that the ability to recognise and stimulate G(i) did not involve the third cytoplasmic loop of M(2). We investigated peptide elements taken from the second intracellular loop of the M(2) receptor for their ability to mediate activation of G(i) and found that a nine amino acid peptide representing the C-terminal sequence, VKRTTKMAG-NH2 (V9G), was capable of inhibiting forskolin-stimulated adenylyl cyclase by up to 18% and could stimulate high affinity GTPase activity of rat brain membranes by 32%. Further, V9G was shown to cause a doubling of the initial rate 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 the second intracellular loop of the muscarinic M(2) receptor that is involved in the selection of a pertussis toxin-sensitive G protein.