β-Arrestin-Dependent Spontaneous α_1a-Adrenoceptor Endocytosis Causes Intracellular Transportation of α-Blockers via Recycling Compartments

Abstract

The antagonist ligand BODIPY-FL-prazosin (QAPB) fluoresces when bound to bovine ␣ 1a -adrenoceptors (ARs). Data indicate that the receptor-ligand complex is spontaneously internalized by ␤-arrestin-dependent endocytosis. Internalization of the ligand did not occur in ␤-arrestin-deficient cells; was blocked or reversed by another ␣ 1 ligand, phentolamine, indicating it to reflect binding to the orthosteric recognition site; and was prevented by blocking clathrin-mediated endocytosis. The ligand showed rapid, diffuse, low-intensity, surface binding, superseded by punctate intracellular binding that developed to equilibrium in 50 to 60 min and was reversible on ligand removal, indicating a dynamic equilibrium. In cells expressing a human ␣ 1a -AR-enhanced green fluorescent protein (EGFP) 2 fusion protein, BODIPY-R-558/568-prazosin (RQAPB) colocalized with the fusion, indicating that the ligand gained access to all compartments containing the receptor, and, conversely, that the receptor has affinity for the ligand at all of these sites. The distribution of QAPB binding sites was similar for receptors with or without EGFP2, validating the fusion protein as an indicator of receptor location. The ligand partially colocalized with ␤-arrestin in recycling and late endosomes, indicating receptor transit without destruction. Organelles containing receptors showed considerable movement consistent with a transportation function. This was absent in ␤-arrestin-deficient cells, indicating that both constitutive receptor internalization and subsequent intracellular transportation are ␤-arrestin-dependent. Calculations of relative receptor number indicate that at steady state, less than 30% of receptors reside on the cell surface and that recycling is rapid. We conclude that ␣ 1a -ARs recycle rapidly by an agonist-independent, constitutive, ␤-arrestin-dependent process and that this can transport "␣-blockers" into cells carrying these receptors.

The structural basis of arrestin-mediated regulation of G-protein-coupled receptors

“…The metabotropic glutamate receptor mGluR1a undergoes extensive constitutive agonist-independent internalization that does not involve arrestins, yet its internalization in response to agonist is selectively mediated by arrestin2 (Dale et al, 2001). In contrast, spontaneous internalization of the α1A-adrenoreceptor is arrestin dependent, and this receptor travels with bound arrestin all the way to the recycling endosomes (Pediani et al, 2005). WT serotonin 5-HT4 receptor internalizes via an arrestin-dependent pathway in colliculi neurons naturally expressing it, as well as in COS-7 and HEK293 cells, but the deletion of the main S/T cluster phosphorylated by GRK2 re-directs the receptor to an arrestin-independent pathway and actually increases the rate of its internalization .…”

Helix-1 of the cAMP-specific phosphodiesterase PDE4A1 regulates its phospholipase-D-dependent redistribution in response to release of Ca2+

“…Where quantification was to be carried out, a Nikon Diaphot inverted microscope was used equipped with a 40ϫ oil-immersion Fluor objective lens (1.3 NA). A monochromator (Optoscan) was used to excite cells and fluorescence emission was detected by a cooled digital charge-coupled device camera (Cool Snap-HQ; Photometrics, Tucson, AZ), as described previously by others (Pediani et al, 2005). Cells were analysed 24-48 hours after transfection.…”

Systems Analysis of the Cardiac Myocyte Hypertrophy Signaling Network

“…In addition to our BODIPY-prazosin data in neonatal cardiac myocytes, recent evidence has shown a large population of α 1 AR's in adult cardiac myocytes [54]. Moreover, expressing α 1a AR's in R-1F cells were predominately found in intracellular organelles [53] and 40% of native α 1 AR's in human smooth muscle cells were intracellular [62]. Prazosin, an αAR antagonist that can act at the sarcolemma and also be transported Functional intracellular receptors have been identified for angiotensin II [63], endothelin-A and -B [64], and β-adrenergic receptors [65] in cardiac myocytes.…”

“…BODIPY-prazosin fluoresces when bound to αAR's and remains effectively non-fluorescent when unbound [61]. Images revealed that the majority of data that showed that expressing α 1a AR's in R-1F cells were predominately found in intracellular organelles, including early and late endosomes [53]. These punctate vesicular structures are also apparent in BODIPY-prazosin images from adult mouse cardiac myocytes [54].…”

“…Recent evidence that αAR's colocalize with intracellular endosomes [53] and the nuclear membrane in adult cardiac myocytes [54] provides a potential mechanism for this intracellular αAR signaling. Therefore, we hypothesized that the concentration dependent reversibility delay is explained by intracellular ligand trapping: internalized PE acting on intracellular αAR's.…”