Differences in the cellular localization and agonist-mediated internalization properties of the alpha(1)-adrenoceptor subtypes

Abstract

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