To identify residues and domains of the peptide hormone pituitary adenylate-cyclase-activating polypeptide (PACAP) that interact with the type I receptor, two photoreactive analogues of PACAP-( 1-27)-peptide were synthesized using solid-phase peptide synthesis. Phe6 or Tyr22 within the PACAP sequence were replaced by p-benzoyl-L-phenylalanine (Bz-Phe) thus creating two PACAP derivatives with a photoreactive amino acid in either the disordered N-terminal or the helical C-terminal part of the peptide. The ligand-binding properties and the efficiencies of these peptide analogues as photolabels were tested for pig brain PACAP receptors.
The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor belongs to the glucagon/secretin/ vasoactive intestinal polypeptide (VIP) receptor family. We mutated and deleted an amino acid residue (E261) which 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 surface of COS-7 cells at nearly the same level and revealed the same high affinity for the agonist PACAP-27. The cAMP contents of COS cells transfected with the E261A, E261Q, and the deletion mutant receptor were 4.6-, 5.7-, and 6.7-fold higher as compared with COS cells transfected with the wild-type 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.z 2000 Federation of European Biochemical Societies.
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