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

摘要

In the present study, we have analyzed a previously identified constitutively active pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptor with a deletion of the single amino acid residue Glu~(261) (Y.-J. Cao, C. Gimpl, F. Fahrenholz, A mutation of second intracellular loop of pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation, FEBS Lett. 469 (2000)). This glutamic acid residue is highly conserved within the second intracellular loop of class II G protein-coupled receptors and may thus be of importance for many members 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 PACl 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 PACl receptor expressed in COS cells. The constitutively active PACl receptor was expressed at the surface of COS-7 cells at the same density as the wild-type receptor. With respect to the different photoreactive PACAP analogues, the labeling specificity was the same for the wild-type versus mutant receptor: ~(125)I-[Lys~(15)(pBz_2)]-PACAP-27 and ~(125)I-[Bpa~(22)]-PACAP-27 were efficiently incorporated into each of the receptors, whereas ~(125)I-[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 α-helical region of the PACAP-27 molecule. However, in comparison with the wild-type PACl receptor, the constitutively active receptor showed a markedly (approx. 6-8-fold) enhanced photoaffinity labeling efficiency in particular of the high glycosylated form. The enzymatically deglycosylated rat PACl receptor was efficiently labeled by photoreactive PACAP analogues. In contrast, nonglycosylated PACl receptors produced by tunicamycin treatment of the transfected COS-7 cells showed a 30-fold lower affinity for PACAP-27 and were capable of signal transduction with 30-50-fold lower potency as compared with the glycosylated PACl receptors.