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

α1D-Adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes

“…Under identical conditions, the cultured cardiac myocytes did exhibit a robust intracellular Ca 2+ signal to α 1 AR activation (Figure 4) due to a different functional receptor subtype, the α 1A -ARs, responsible for this effect (see refs 24, and 31-33). Additionally, α 1A -ARs in the cultured cardiac myocytes were found both intracellularly and at the cell membrane distributions, whereas α 1D -ARs were most frequently concentrated intracellularly in cultured VSMCs ( Figures 6A and B), consistent with existing research [7][8][9][10]31] . Interestingly, similar α 1 AR distribution patterns, α 1 adrenergic-sensitive cell shortening and [Ca 2+ ] i increases (as manifested in the cardiomyocytes) were clearly observable in most of the freshly isolated aortic myocytes, but disappeared completely in VSMCs cultured ≥ 2 days (Figures 3 Table 1).…”

“…Although all three subtypes are coupled to GPCR, they differ in their tissue distributions, affinities to ligands, and apparent efficiencies for intracellular couplings [6,7] . To date, the clearest differences among the α 1 AR subtypes are in their subcellular localizations: α 1A -ARs and α 1B -ARs are primarily located at the plasma membrane while the majority of α 1D -ARs are found accumulated within the intracellular compartments of both recombinant and native cells [7][8][9][10] . Additionally, divergent factors or mechanisms have been found to differentially regulate the subtypes in regards of the receptor internalization/trafficking recycling, expression, and sensitivity of the signaling and effector molecules that they α 1D -Adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes 1586 www.nature.com/aps Fan LL et al Acta Pharmacologica Sinica npg are coupling, rendering a fine tuning for the α 1 -adrenergic responsiveness to different biological signals and for adapting cells to changes in the internal milieu and to overall homeostasis [2,6,[10][11][12] .…”

Impaired Feedback Regulation of the Receptor Activity and the Myofilament Ca2+ Sensitivity Contributes to Increased Vascular Reactiveness after Subarachnoid Hemorrhage

“…The phosphorylation of receptor by G proteincoupled receptor kinases and the binding of barrestin to receptor contribute to the rapid termination of receptor signaling, while the endocytotic internalization and the lysosomal degradation also contribute to the subsequent termination (Drake et al, 2006;Moore et al, 2007). The relative contribution of each step and precise molecular mechanism varies depending on the type of receptor (Abe et al, 2000;Chalothorn et al, 2002;Freedman et al, 1997). Therefore, the step common to ET A receptor, PAR 1 , and a 1 -adrenoceptor is considered to be impaired in SAH, and the molecule(s) positively or negatively involved in such a step may be up-or downregulated in the cerebral artery after SAH.…”

Stress Impairs α1A Adrenoceptor-Mediated Noradrenergic Facilitation of GABAergic Transmission in the Basolateral Amygdala

“…In other brain regions, repeated stress reduces mRNA levels of a 1 adrenoceptors (Miyahara et al, 1999). Adrenergic receptors desensitize or undergo downregulation following prolonged exposure to the agonist (Yang et al, 1999;Chalothorn et al, 2002). Thus, during stress exposure, excessive release of NE in the amygdala (Galvez et al, 1996;Quirarte et al, 1998;Tanaka et al, 2000) may be responsible for the impairment of the a 1A adrenoreceptor function.…”