B. Deslauriers et al., N-glycosylation requirements for the AT(1a) angiotensin II receptor delivery to the plasma membrane, BIOCHEM J, 339, 1999, pp. 397-405
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) angiote
nsin II (AII) receptor. Glycosylation-site suppression was carried out by s
ite-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-termi
nal 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-glycosyl
ation-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 immunocytochemica
l cellular localization of the various receptors transiently expressed in C
OS-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-glycosylat
ion sites was not (or was very inefficiently) expressed at the plasma membr
ane and accumulated inside the eel at the perinuclear zone; (iii) the prese
rvation of two sites allowed normal receptor delivery to the plasma membran
e, the presence of only Asn(176) ensuring a behaviour similar to that of th
e wild-type receptor; and (iv) all expressed receptors displayed unchanged
pharmacological properties (K-d for I-125-sarcosine(1)-AII; sarcosine(1)-AI
I-induced inositol phosphate production). These results demonstrate that N-
glycosylation is required for the AT(1) receptor expression. They are discu
ssed in the light of current knowledge of membrane-protein maturation and f
uture prospects of receptor overexpression for structural studies.