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 α1B-AR/GFP fluorescence was detected predominantly on the cell surface. Stimulation of the α1B-AR with phenylephrine led to an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and promoted rapid α1B-AR/GFP internalization. Long-term exposure (15 h) to phenylephrine resulted in desensitization of the α1B-AR-mediated activation of ERK1/2 phosphorylation. α1A-AR/GFP fluorescence was detected not only on the cell surface but also intracellularly. The rate of internalization of the cell surface population α1A-AR/GFPs was slower than that seen for the α1B-AR. Agonist exposure also resulted in desensitization of the α1A-AR-mediated increase in ERK1/2 phosphorylation. The α1D-AR/GFP fluorescence was detected mainly intracellularly, and this localization was unaffected by exposure to phenylephrine. Phenylephrine treatment of α1D-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 α1A- or α1B-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 α1A- and α1B-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.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|