Differences in the Cellular Localization and Agonist-Mediated Internalization Properties of the α1-Adrenoceptor Subtypes

• Published in: Molecular pharmacology Vol. 61; no. 5; p. 1008
• Main Authors: Dan Chalothorn, Dan F. McCune, Stephanie E. Edelmann, Mary L. Garcı́a-Cazarı́n, Gozoh Tsujimoto, Michael T. Piascik
• Format: Journal Article
• Language: English
• Published: American Society for Pharmacology and Experimental Therapeutics 01.05.2002
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.61.5.1008

The cellular localization, agonist-mediated internalization, and desensitization properties of the α 1 -adrenoceptor (α 1 -AR) subtypes conjugated with green fluorescent protein (α 1 -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 α 1 -AR/GFP fusion proteins are functional, that there are differences in the cellular distribution and agonist-mediated internalization between the α 1 -ARs, and that agonist-mediated α 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 α 1 -ARs.