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Titolo:
A constitutively active pituitary adenylate cyclase activating polypeptide(PACAP) type I receptor shows enhanced photoaffinity labeling of its highly glycosylated form
Autore:
Cao, YJ; Gimpl, G;
Indirizzi:
Univ Mainz, Inst Biochem, D-55099 Mainz, Germany Univ Mainz Mainz Germany D-55099 z, Inst Biochem, D-55099 Mainz, Germany
Titolo Testata:
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
fascicolo: 1, volume: 1548, anno: 2001,
pagine: 139 - 151
SICI:
0167-4838(20010709)1548:1<139:ACAPAC>2.0.ZU;2-G
Fonte:
ISI
Lingua:
ENG
Soggetto:
VASOACTIVE-INTESTINAL-PEPTIDE; FUNCTIONAL EXPRESSION; TISSUE DISTRIBUTION; MOLECULAR-CLONING; BINDING-SITES; HORMONE; DOMAIN; BRAIN; CELLS; VIP;
Keywords:
pituitary adenylate cyclase activating polypeptide; photoaffinity labeling; peptide hormone receptor; glycosylation; mutation;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
43
Recensione:
Indirizzi per estratti:
Indirizzo: Cao, YJ Schering AG, Res Labs, Dept Expt Oncol, Mullerstr 178, D-13342 Berlin, Germany Schering AG Mullerstr 178 Berlin Germany D-13342 Berlin, Germany
Citazione:
Y.J. Cao e G. Gimpl, "A constitutively active pituitary adenylate cyclase activating polypeptide(PACAP) type I receptor shows enhanced photoaffinity labeling of its highly glycosylated form", BBA-PROT ST, 1548(1), 2001, pp. 139-151

Abstract

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 Glu261 (Y.-J. Cao. G. Gimpl. F. Fahrenholz, A mutation of second intracellular loopof pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation. FEES Lett. 469 11000)). This glutamicacid residue is highly conserved within the second intracellular loop of class IT G protein-coupled receptors and may thus be of importance for many members of this receptor class. To explore the molecular characteristics ofthis mutant receptor, we performed photoaffinity labeling using previouslydefined photoreactive PACAP analogues. In COS cells, the PAC1 receptor wasexpressed in two differently glycosylated forms: a M-r 75 000 and a M-r 55000 form. According to partial deglycosylation, at least three carbohydrate chains may exist in the rat PAC1 receptor expressed in COS cells. The constitutively 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 different 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 receptors, 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 toTyr(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) enhanced photoaffinity labeling efficiency in particular of the high glycosylated form. The enzymatically deglycosylated rat PAC1 receptor was efficiently labeled by photoreactive PACAP analogues. In contrast, nonglycosylatedPAC1 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 PAC 1 receptors. (C) 2001 Published by Elsevier Science B.V.

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Documento generato il 29/03/20 alle ore 12:08:33