Abstract

The potential role of dimerization in controlling the expression and pharmacological properties of α₁-adrenoceptor subtypes was examined using coimmunoprecipitation of epitope-tagged receptors. Human α₁-adrenoceptor subtypes (α_1A, α_1B, α_1D) were tagged at their amino-termini with Flag or hemagglutinin epitopes and transfected into human embryonic kidney 293 cells. Homodimerization of all three subtypes was observed by coimmunoprecipitation of receptors with different tags and was not altered by norepinephrine treatment. Heterodimer formation between hemagglutinin-tagged α_1B-adrenoceptors and Flag-tagged α_1A- or α_1D-adrenoceptors was also observed. However, no α_1A/α_1D-adrenoceptor heterodimers were observed, suggesting that dimerization is subtype-specific. The extent of heterodimerization was also unaltered by norepinephrine treatment. α₁-Adrenoceptor truncation mutants lacking carboxyl or amino-terminal sequences formed homo- and heterodimers similarly to full-length receptors, suggesting that these domains play little or no role in dimerization. Biotinylation with a membrane-impermeable agent showed that monomers and homo- and hetero-oligomers of all three subtypes are expressed on the cell surface. Radioligand binding studies showed that heterodimerization did not alter the affinity of α₁-adrenoceptors for norepinephrine, prazosin, or subtype-selective antagonists, suggesting that dimerization does not result in pharmacologically distinct subtypes. However, coexpression of α_1B-adrenoceptors significantly increased both binding site density and protein expression of α_1A- and α_1D-adrenoceptors, and increased cell surface expression of α_1D-adrenoceptors, suggesting a functional role for heterodimerization. Conversely, coexpression of α_1A-with α_1D-adrenoceptors, which did not heterodimerize, had no effect on receptor density or protein. These studies demonstrate subtype-selective heterodimerization of α₁-adrenoceptors, which does not change their pharmacological properties but seems to have functional consequences in regulating receptor expression and trafficking.