N-terminal truncation of human α 1D-adrenoceptors increases expression of binding sites but not protein

Abstract

The role of the N-terminus of human α 1D-adrenoceptors was examined by deleting the first 79 amino acids (Δ 1–79) and epitope-tagging to facilitate immunoprecipitation and detection. Following transfection into HEK293 cells, 6- to 13-fold increases in the density of specific [ 125I]BE 2254 binding sites were observed for both tagged and untagged Δ 1–79α 1D- compared to full-length α 1D-adrenoceptors, while agonist and antagonist affinities remained unchanged. In contrast, immunoprecipitation of tagged receptors showed that full-length α 1D-adrenoceptor protein was at least twice as abundant as Δ 1–79α 1D-adrenoceptor protein. Photoaffinity labelling with [ 125I]arylazidoprazosin showed much more intense labelling of tagged Δ 1–79α 1D- than of full-length α 1D-adrenoceptors. Substantial N-linked glycosylation of tagged Δ 1–79α 1D-adrenoceptors was observed, although full-length α 1D-adrenoceptors contain two consensus glycosylation sites but are not glycosylated. These results suggest that N-terminal truncation of α 1D-adrenoceptors enhances processing of a binding competent form in HEK293 cells; and show a clear dissociation between abundance of receptor protein and density of receptor binding sites.