Activation of Gi protein by peptide structures of the muscarinic M2 receptor second intracellular loop.

The muscarinic M2 receptor that normally couples via Gi to inhibit adenylyl cyclase was made to couple to Gs by exchange of its third intracellular loop for the comparable domain of the beta 2-adrenoceptor. In HeLa cells transfected with the recombinant M2 beta i-3 cDNA, the chimaeric receptor showed carbachol-mediated activation of adenylyl cyclase (EC50 = 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 M2 beta i-3 receptor was able to couple to both Gi and Gs, and that the ability to recognise and stimulate Gi did not involve the third cytoplasmic loop of M2. We investigated peptide elements taken from the second intracellular loop of the M2 receptor for their ability to mediate activation of Gi 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 [35S]GTP gamma S binding to purified bovine brain Gi/Go in reconstituted phospholipid vesicles. These data identify a domain on the second intracellular loop of the muscarinic M2 receptor that is involved in the selection of a pertussis toxin-sensitive G protein.