Role of charged amino acids conserved in the vasoactive intestinal polypeptide/secretin family of receptors on the secretin receptor functionality

The secretin receptor is a member of a large family of G-protein-coupled re ceptors that recognize polypeptide hormone and/or neuropeptides. Charged, c onserved residues might play a key role in their function, either by intera cting with the ligand or by stabilizing the receptor structure. Of the four charged amino acids that are conserved in the whole secretin receptor fami ly, D49 and R83 (in the N-terminal domain) were probably important for the secretin receptor structure: replacement of D49 by H or R and of R83 by D s everely reduced both the maximal response to secretin and its potency. No f unctional secretin receptor could be detected after replacement of R83 by L . Mutation of D49 to E, A, or N had no effect or reduced 5-fold the potency of secretin. The highly conserved positive charges found at the extracellu lar ends of TM III (K194) and IV (R255) were important for the secretin rec eptor function, as K194 mutation to A or Q and R255 mutation to Q or D decr eased the secretin's affinity 15- to 1000-fold, respectively. Six extracell ular charged residues are conserved in closely related receptors but not in the whole family. K121 (TM I) and R277 (TM V) were not important for funct ional secretin receptor expression. D174 (TM II) was necessary to stabilize the active receptor structure: the D174N mutant receptors were unable to s timulate normally the adenylate cyclase in response to secretin, and functi onal D174A receptors could not be found. Mutation of R255, E259 (second ext racellular loop), and E351 (third extracellular loop) to uncharged residues reduced only 10- to 100-fold the secretin potency without changing its eff icacy: these residues either stabilized the active receptor conformation or formed hydrogen rather than ionic bonds with secretin. Mutation of K121 (T M I) to Q or L and of R277 (TM V) to E or Q did not affect the receptor fun ctional properties. (C) 1999 Elsevier Science Inc. All rights reserved.