Cellular Trafficking of Human α1a-Adrenergic Receptors Is Continuous and Primarily Agonist-Independent

Abstract

␣ 1a -Adrenergic receptors (␣ 1a ARs) are present intracellularly and at the cell surface in cultured and natural cell models, where they are subject to agonist-mediated desensitization and internalization. To explore ␣ 1a AR trafficking, a hemagglutinin (HA)-tagged ␣ 1a AR/enhanced green fluorescent protein (EGFP) fusion protein was expressed in rat-1 fibroblasts and tracked by EGFP fluorescence and antibody labeling of surface receptors. Confocal analysis of antibody-labeled surface receptors revealed unexpected constitutive internalization in the absence of agonist stimulation. In partial agreement, the inverse agonist prazosin also caused a modest 20 Ϯ 2% increase in surface receptor levels, suggesting a partial block of constitutive internalization caused by decreased basal activation. However, prazosin was unable to prevent internalization of antibody-tagged surface receptors observed by confocal microscopy or cause obvious redistribution of intracellular receptor to the surface, suggesting that the ␣ 1a AR is internalizing even in a basalinactive state. In contrast to the ␣ 1a AR, surface labeling of an HA-tagged ␣ 1b -EGFP fusion protein did not result in any apparent constitutive internalization. Constitutive internalization of the ␣ 1a AR seems to occur alongside reversible agonist-induced internalization, and both seem to involve clathrin-mediated endocytosis but not degradation in lysozymes. Surface receptor density must be maintained by recycling, because the protein synthesis inhibitor cycloheximide has no effect on total or surface receptor density in agonist-treated or untreated cells for 6 h. Constitutive agonist-independent trafficking of ␣ 1a ARs may provide a novel mechanism by which an internal pool of ␣ 1a ARs are maintained and recycled to allow continuous agonist-induced signaling.

Differential Agonist-Mediated Internalization of the Human 5-Hydroxytryptamine 7 Receptor Isoforms

“…The constitutive internalization of the 5-HT 7(d) isoform is distinct from the signaling pathway as untreated cells show no cAMP-mediated response and a 5-HT 7 receptor antagonist inhibits agonist-mediated reporter gene activity in a dosedependent manner. Although no such trafficking differences have been reported among the serotonin receptors, similar trafficking patterns have been reported for the ␣ 1 adrenergic receptors (Hirasawa et al, 1997;Chalothorn et al, 2002;Hague et al, 2003;Morris et al, 2004). However, these ␣ 1 adrenergic receptors are transcribed from three separate genes.…”

Constitutive Activation Drives Compartment-Selective Endocytosis and Axonal Targeting of Type 1 Cannabinoid Receptors

“…A growing body of evidence indicates that this behavior is likely a general phenomenon for constitutively active GPCRs (Whistler et al, 2002;Fourgeaud et al, 2003;Leterrier et al, 2004;Marion et al, 2004;Morris et al, 2004). However, these results were obtained in heterologous expression systems, so the physiological relevance of this phenomenon remains unknown.…”

“…Antagonist ligands that inhibit this basal activation by stabilizing inactive receptor conformations are termed inverse agonists (Milligan and Bond, 1997), whereas true "neutral" antagonists inhibit binding of both agonists and inverse agonists without favoring the equilibrium between active or inactive conformations (Kenakin, 2004). Interesting consequences of constitutive activity were reported recently for GPCRs such as the type 1 cannabinoid receptor (CB1R), the 5-HT 2C serotonin receptor, or the ␣ 1a adrenergic receptor (Leterrier et al, 2004;Marion et al, 2004;Morris et al, 2004). These constitutively active GPCRs are constitutively endocytosed from the plasma membrane and, thus, exhibit a mostly intracellular (endosomal) localization at steady-state.…”

The effect of angiotensin II on intracellular pH is mediated by AT1 receptor translocation

“…Cells were incubated for 5 h at 37°C with 5% CO 2 and then cultured for 24 h in DMEM supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 g/ml streptomycin, and 2 mmol/l L-glutamine. The individual cell lines that stably expressed the recombinant hAT 1/pEGFP-N1 were selected with geneticin (G418 1 mg/ml) and used for all experiments (passages [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19].…”