Abstract

The cellular localization, agonist-mediated internalization, and desensitization properties of the α₁-adrenoceptor (α₁-AR) subtypes conjugated with green fluorescent protein (α₁-AR/GFP) were assessed using real-time imaging of living, transiently transfected human embryonic kidney (HEK) 293 cells. The α_1B-AR/GFP fluorescence was detected predominantly on the cell surface. Stimulation of the α_1B-AR with phenylephrine led to an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and promoted rapid α_1B-AR/GFP internalization. Long-term exposure (15 h) to phenylephrine resulted in desensitization of the α_1B-AR-mediated activation of ERK1/2 phosphorylation. α_1A-AR/GFP fluorescence was detected not only on the cell surface but also intracellularly. The rate of internalization of the cell surface population α_1A-AR/GFPs was slower than that seen for the α_1B-AR. Agonist exposure also resulted in desensitization of the α_1A-AR-mediated increase in ERK1/2 phosphorylation. The α_1D-AR/GFP fluorescence was detected mainly intracellularly, and this localization was unaffected by exposure to phenylephrine. Phenylephrine treatment of α_1D-AR/GFP expressing cells increased ERK1/2 phosphorylation. However, this increase was not significant. Cotransfection with β-arrestin 1 did not increase the rate or extent of agonist-stimulated α_1A- or α_1B-AR/GFP internalization. However, a dominant-negative form of the β-arrestin 1, β-arrestin 1 (319–418), blocked agonist-mediated internalization of both the α_1A- and α_1B-ARs. These data show that transfected α₁-AR/GFP fusion proteins are functional, that there are differences in the cellular distribution and agonist-mediated internalization between the α₁-ARs, and that agonist-mediated α₁-AR internalization is dependent on arrestins and can be desensitized by long-term exposure to an agonist. These differences could contribute to the diversity in physiologic responses regulated by the α₁-ARs.