Pediani John D, Colston Janet F, Caldwell Darren, Milligan Graeme, Daly Craig J, McGrath John C
Autonomic Physiology Unit, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Mol Pharmacol. 2005 Apr;67(4):992-1004. doi: 10.1124/mol.104.008417. Epub 2004 Dec 30.
The antagonist ligand BODIPY-FL-prazosin (QAPB) fluoresces when bound to bovine alpha(1a)-adrenoceptors (ARs). Data indicate that the receptor-ligand complex is spontaneously internalized by beta-arrestin-dependent endocytosis. Internalization of the ligand did not occur in beta-arrestin-deficient cells; was blocked or reversed by another alpha(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 The ligand showed rapid, diffuse, low-intensity, surface binding, superseded by punctate intracellular binding that developed to equilibrium in 50 to 60 min and was reversible on ligand removal, indicating a dynamic equilibrium.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 alpha(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 beta-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 beta-arrestin-deficient cells, indicating that both constitutive receptor internalization and subsequent intracellular transportation are beta-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 alpha(1a)-ARs recycle rapidly by an agonist-independent, constitutive, beta-arrestin-dependent process and that this can transport "alpha-blockers" into cells carrying these receptors.
拮抗剂配体BODIPY-FL-哌唑嗪（QAPB）与牛α(1a)-肾上腺素能受体（ARs）结合时会发出荧光。数据表明，受体-配体复合物通过β-抑制蛋白依赖性内吞作用自发内化。在缺乏β-抑制蛋白的细胞中，配体不会发生内化；另一种α(1)配体酚妥拉明可阻断或逆转这种内化，表明这反映了与正构识别位点的结合；并且通过阻断网格蛋白介导的内吞作用可阻止这种内化。该配体表现出快速、弥散、低强度的表面结合，随后被点状的细胞内结合所取代，这种结合在50至60分钟内达到平衡，并且在去除配体后是可逆的，表明存在动态平衡。在表达人α(1a)-AR-增强型绿色荧光蛋白（EGFP）2融合蛋白的细胞中，BODIPY-R-558/568-哌唑嗪（RQAPB）与该融合蛋白共定位，表明该配体能够进入所有含有受体的区室，反之，受体在所有这些位点都对该配体具有亲和力。对于有或没有EGFP2的受体，QAPB结合位点的分布相似，这验证了该融合蛋白可作为受体定位的指标。该配体在回收型和晚期内体中与β-抑制蛋白部分共定位，表明受体在转运过程中未被破坏。含有受体的细胞器表现出与运输功能一致的大量移动。在缺乏β-抑制蛋白的细胞中则不存在这种情况，这表明组成型受体内化和随后的细胞内运输均依赖于β-抑制蛋白。相对受体数量的计算表明，在稳态下，不到30%的受体位于细胞表面，并且回收过程很快。我们得出结论，α(1a)-ARs通过一种不依赖激动剂、组成型、β-抑制蛋白依赖性的过程快速循环，并且这可以将“α阻滞剂”转运到携带这些受体的细胞中。
