N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane

Abstract

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.

Angiotensin II Type 1 Receptor Mechanoactivation Involves RGS5 (Regulator of G Protein Signaling 5) in Skeletal Muscle Arteries

“…The AT 1 R has also been shown to undergo post-translational glycosylation 31 and the glycosylated AT 1 R is predicted to have a higher molecular weight. 33 In the present study rat kidney (used as a positive control) showed the ≈58 kDa glycosylated band, consistent with previous reports 34 and this higher molecular weight band was also detected in the cultured cremaster myocytes (Figure S5A, Left Column). As an index of specificity Western blotting was performed after pre-treating the antibody with a blocking peptide/antigen-directed against the AT 1 R (Figure S5A, Right Column).…”

Molecular regulation of lysophosphatidic acid receptor 1 trafficking to the cell surface

“…N-glycosylation regulates GPCR structure and maturation [34–36]. Consistent with the findings from Versano et al [26], a prominent broad band of LPA1-V5 at ~60 kDa was shifted to ~40 kDa after treatment with PNGase F, an endoglycosidase to remove N-linked glycans (Fig.…”

Lack of Specificity of Commercial Antibodies Leads to Misidentification of Angiotensin Type 1 Receptor Protein

“…A single band at ~51 kDa was present in all samples including the mock-transfected cells and likely represents endogenous His proteins expressed by HEK cells (marked with an asterisk on Fig 5b). Nevertheless, the ~39 kDa band is consistent with the non-glycosylated form of the AT 1 R 12 even though one may question the apparent molecular weight of AT 1 R “monomer” because this band appears to be slightly smaller than the predicted 41 kDa mass of this protein. This small apparent difference in size could be accounted for by an altered migration velocity due to the positively charged histidines added to the carboxyl-terminal tail.…”

“…Since both AT 1 R isoforms undergo post-translational glycosylation, a single ~41 kDa band should represent the non-glycosylated AT 1A or AT 1B receptor whereas higher molecular bands would be expected for the glycosylated forms. 12 …”

“…Accordingly, using a plasmid expressing the AT 1 R tagged to a myc epitope, Deslauriers and colleagues reported massive AT 1 R glycosylation in transfected COS-7 cells. 12 In that study, the molecular size of the glycosylated AT 1 R form was estimated to be ~100-150kDa. Since the degree of glycosylation of proteins is a tissue-specific process, it is difficult to predict the molecular mass of these receptors under different tissues or experimental conditions and published data clarifying this issue are lacking.…”