A constitutively active pituitary adenylate cyclase activating polypeptide(PACAP) type I receptor shows enhanced photoaffinity labeling of its highly glycosylated form

In the present study, we have analyzed a previously identified constitutive ly active pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptor with a deletion of the single amino acid residue Glu261 (Y .-J. Cao. G. Gimpl. F. Fahrenholz, A mutation of second intracellular loop of pituitary adenylate cyclase activating polypeptide type I receptor confe rs constitutive receptor activation. FEES Lett. 469 11000)). This glutamic acid residue is highly conserved within the second intracellular loop of cl ass IT G protein-coupled receptors and may thus be of importance for many m embers of this receptor class. To explore the molecular characteristics of this mutant receptor, we performed photoaffinity labeling using previously defined photoreactive PACAP analogues. In COS cells, the PAC1 receptor was expressed in two differently glycosylated forms: a M-r 75 000 and a M-r 55 000 form. According to partial deglycosylation, at least three carbohydrate chains may exist in the rat PAC1 receptor expressed in COS cells. The cons titutively active PAC1 receptor was expressed at the surface of COS-7 cells at the same density as the wild-type receptor. With respect to the differe nt photoreactive PACAP analogues, the labeling specificity was the same for the wild-type versus mutant receptor. I-125-[Lys(15)(pBz(2))]-PACAP-27 and I-125-[Bpa(22)]-PACAP-27 were efficiently incorporated into each of the re ceptors, whereas I-125-[Bpa(6)]-PACAP-27 labeled each of the receptors only to a negligible extent. This suggests that both receptors have the same or at least a very similar hormone binding site which is in close contact to Tyr(22) and Lys(15) located in the carboxy-terminal a-helical region of the PACAP-27 molecule. However, in comparison with the wild-type PAC1 receptor . the constitutively active receptor showed a markedly (approx. 6-8-fold) e nhanced photoaffinity labeling efficiency in particular of the high glycosy lated form. The enzymatically deglycosylated rat PAC1 receptor was efficien tly labeled by photoreactive PACAP analogues. In contrast, nonglycosylated PAC1 receptors produced by tunicamycin treatment of the transfected COS-7 c ells showed a 30-fold lower affinity for PACAP-27 and were capable of signa l transduction with 30-50-fold lower potency as compared with the glycosyla ted PAC 1 receptors. (C) 2001 Published by Elsevier Science B.V.