N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane.
The purpose of this work was to investigate the role of N-glycosylation in the expression and pharmacological properties of the the rat AT1a angiotensin II (AII) receptor. Glycosylation-site suppression was carried out by site-directed mutagenesis (Asn-->Gln) of Asn176 and Asn188 (located on the second extracellular loop) and by the removal of Asn4 at the N-terminal end combined with the replacement of the first four amino acids by a 10 amino acid peptide epitope (c-Myc). We generated seven possible N-glycosylation-site-defective mutants, all tagged at their C-terminal ends with the c-Myc epitope. This double-tagging strategy, associated with photoaffinity labelling, allowed evaluation of the molecular masses and immunocytochemical cellular localization of the various receptors transiently expressed in COS-7 cells. We showed that: (i) each of the three N-glycosylation sites are utilized in COS-7 cells; (ii) the mutant with three defective N-glycosylation sites was not (or was very inefficiently) expressed at the plasma membrane and accumulated inside the cell at the perinuclear zone; (iii) the preservation of two sites allowed normal receptor delivery to the plasma membrane, the presence of only Asn176 ensuring a behaviour similar to that of the wild-type receptor; and (iv) all expressed receptors displayed unchanged pharmacological properties (Kd for 125I-sarcosine1-AII; sarcosine1-AII-induced inositol phosphate production). These results demonstrate that N-glycosylation is required for the AT1 receptor expression. They are discussed in the light of current knowledge of membrane-protein maturation and future prospects of receptor overexpression for structural studies.
Deslauriers
B
B
Laboratoire de Médecine Moléculaire, Centre Hospitalier de l'Université de Montréal, 3840 rue St Urbain, Montréal, Quebec, H2W 158, Canada.
Ponce
C
C
Lombard
C
C
Larguier
R
R
Bonnafous
J C
JC
Marie
J
J
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002241:Carbohydrates / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D016296:Mutagenesis; D051381:Rats; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D002241:Carbohydrates; D017931:DNA Primers; D007295:Inositol Phosphates; D011945:Receptors, Angiotensin; D000804:Angiotensin II