Krupnick J G, Gurevich V V, Benovic J L
Department of Biochemistry, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1997 Jul 18;272(29):18125-31. doi: 10.1074/jbc.272.29.18125.
Quenching of phototransduction in retinal rod cells involves phosphorylation of photoactivated rhodopsin by the enzyme rhodopsin kinase followed by binding of the protein arrestin. Although it has been proposed that the mechanism of arrestin quenching of visual transduction is via steric exclusion of transducin binding to phosphorylated light-activated rhodopsin (P-Rh*), direct evidence for this mechanism is lacking. In this study, we investigated both the role of rhodopsin phosphorylation in modulating its interaction with arrestin and transducin and the proposed binding competition between arrestin and transducin for P-Rh*. While the beta-adrenergic receptor kinase promotes significant arrestin binding to rhodopsin at a phosphorylation stoichiometry of >/=2 mol/mol, rhodopsin kinase promotes arrestin binding at a stoichiometry of approximately 0.9 mol/mol. Moreover, while beta-adrenergic receptor kinase phosphorylation of rhodopsin only modestly decreases transducin binding and activation, rhodopsin kinase phosphorylation of rhodopsin significantly decreases transducin binding and activation. Finally, arrestin competes effectively with transducin for binding to P-Rh* (50% inhibition at approximately 1:1 molar ratio of arrestin:transducin) but has no effect on transducin binding to nonphosphorylated light-activated rhodopsin (Rh*), paralleling the functional inhibition by arrestin on P-Rh*-stimulated transducin activation (50% inhibition at approximately 1.7:1 molar ratio of arrestin:transducin). These results demonstrate that a major role of rhodopsin phosphorylation is to promote high-affinity arrestin binding and decrease transducin binding thus allowing arrestin to effectively compete with transducin for binding to photoactivated rhodopsin.
视网膜视杆细胞中光转导的淬灭涉及视紫红质激酶对光激活视紫红质的磷酸化，随后是抑制蛋白的结合。尽管有人提出抑制蛋白淬灭视觉转导的机制是通过空间位阻阻止转导素与磷酸化的光激活视紫红质（P-Rh*）结合，但缺乏这一机制的直接证据。在本研究中，我们调查了视紫红质磷酸化在调节其与抑制蛋白和转导素相互作用中的作用，以及抑制蛋白和转导素对P-Rh的拟结合竞争。虽然β-肾上腺素能受体激酶在磷酸化化学计量比≥2摩尔/摩尔时能促进抑制蛋白与视紫红质的显著结合，但视紫红质激酶在化学计量比约为0.9摩尔/摩尔时促进抑制蛋白结合。此外，虽然视紫红质的β-肾上腺素能受体激酶磷酸化仅适度降低转导素的结合和激活，但视紫红质的视紫红质激酶磷酸化显著降低转导素的结合和激活。最后，抑制蛋白能有效地与转导素竞争结合P-Rh（在抑制蛋白与转导素摩尔比约为1:1时50%抑制），但对转导素与非磷酸化的光激活视紫红质（Rh*）的结合没有影响，这与抑制蛋白对P-Rh*刺激的转导素激活的功能抑制情况平行（在抑制蛋白与转导素摩尔比约为1.7:1时50%抑制）。这些结果表明，视紫红质磷酸化的一个主要作用是促进高亲和力的抑制蛋白结合并减少转导素结合，从而使抑制蛋白能够有效地与转导素竞争结合光激活的视紫红质。
