A mutation in the second intracellular loop of the pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation

The pituitary adenylate cyclase activating polypeptide (PACAP) type I recep tor belongs to the glucagon/secretin/ vasoactive intestinal polypeptide (VI P) receptor family. We mutated and deleted an amino acid residue (E261) whi ch is located within the second intracellular loop of the rat PACAP type I receptor and which is highly conserved among the receptor family. The wild- type receptor and the mutant receptors were efficiently expressed at the su rface of COS-7 cells at nearly the same level and revealed the same high af finity for the agonist PACAP-27, The cAMP contents of COS cells transfected with the E261A, E261Q, and the deletion mutant receptor mere 4.6-, 5.7-, a nd 6.7-fold higher as compared with COS cells transfected with the wild-typ e receptor. Thus, all the mutant PACAP receptors were constitutively active , The data suggest that the glutamic acid in the second intracellular loop of the PACAP receptor may be a key residue to constrain the receptor in the inactive conformation with respect to its coupling to G(s) proteins. (C) 2 000 Federation of European Biochemical Societies.