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However, introduction of the additional Ser210->Tyr point mutation into this chimeric construct (resulting in m2-Y-Ci3) led to a mutant receptor which, similar to m2-i2 and m2-Ni3, gained coupling to PI turnover (Fig. 7). This result suggested that the N-terminus of the i3 loop (where m3Tyr254 is located) acts in a cooperative fashion with a region at the C-terminus of the same loop to allow proper recognition of Gq/ll proteins. Following these initial results, detailed site-directed mutagenesis studies were carried out to identify individual amino acids within the i2 loop and the C-terminal segment of the i3 loop of the m3 mAChR required for efficient coupling to Gq [39].

36 RECEPTOPJG PROTEIN COUPLING Characteristically, each GPCR can couple only to a limited set of the many structurally similar G proteins expressed in a cell [23]. The same is also true for the individual members of the mAChR family: the ml, m3, and m5 mAChRs are known to preferentially couple to G proteins of the Gq/ll family [2,25,27] which mediate the activation of different isoforms of PLC~ (biochemical response: PI hydrolysis) [24,26,27]. The m2 and m4 mAChRs, on the other hand, are selectively linked to G proteins of the Gi]o class [25,28] which, at a biochemical level, mediate the inhibition of adenylyl cyclase [27].

Proc Natl Acad Sci USA 1991; 88: 11564-11568. Jesaitis AJ. Comments Mol Cell Biophys 1992; 8: 97-114. Jesaitis AJ, Erickson RW, Klotz K-N, Bommakanti RK, Siemsen DW. J Immunol 1993; 151: 5653-5665. den Hartigh JC, van Bergen en Henegouwen PMP, Verkleij AJ, Boonstra J. J Cell Biol 1992; 119: 349-355. Klotz K-N, Jesaitis AJ. Biochem Pharmacol 1994; 48: 1297-1300. Tardif M, Mery L, Brouchon L, Boulay F. J Immunol 1993; 150: 3534-3545. Ali H, Richardson RM, Tomhave ED, Didsbury JR, Snyderman R. J Biol Chem 1993; 268: 24247-24254.

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