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 AT(1a) angiotensin II (AII) receptor. Glycosylation-site suppression was carried out by site-directed mutagenesis (Asn --> Gln) of Asn(176) and Asn(188) (located on the second extracellular loop) and by the removal of Asn(4) 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 eel at the perinuclear zone; (iii) the preservation of two sites allowed normal receptor delivery to the plasma membrane, the presence of only Asn(176) ensuring a behaviour similar to that of the wild-type receptor; and (iv) all expressed receptors displayed unchanged pharmacological properties (K-d for I-125-sarcosine(1)-AII; sarcosine(1)-AII-induced inositol phosphate production). These results demonstrate that N-glycosylation is required for the AT(1) receptor expression. They are discussed in the light of current knowledge of membrane-protein maturation and future prospects of receptor overexpression for structural studies.