Abstract
Purpose: :
To determine possible role of rhodopsin dimerization in arrestin1 binding.
Methods: :
Arrestin1 binding to light-activated phosphorhodopsin (P-Rh*) in native disc membranes and reconstituted into nanodiscs at one molecule per disc was compared.
Results: :
We measured direct binding of radiolabeled arrestin1 to the same preparation of P-Rh* in native disc membranes and reconstituted into nanodiscs at one molecule per disc, varying P-Rh* from 0.02 to 0.3 ug/assay. Both preparations yielded similar binding at the highest P-Rh*, but with decreasing P-Rh* concentration monomeric rhodopsin in nanodiscs progressively outperformed P-Rh* in native membranes (likely due to kinetic advantage of better dispersed rhodopsin in nanodiscs). Arrestin1 affinity (Kd app) for monomeric P-Rh* was ~ 28nM, which is very close to ~ 25nM estimated by Dr. Hofmann’s lab using extra-Meta II assay in native discs. Arrestin1 binding to P-Rh* in nanodiscs with pure phosphatidyl-choline (PC) is low; it increases with the proportion of phosphatidyl-serine (PS). PC:PS ratio in the disc affects arrestin1 on- and off-rates; the affinity (Kd app) reaches plateau at ~30% PS, in agreement with ~20% of PS in native discs. We estimated the stoichiometry of arrestin1 binding to monomeric P-Rh* by incubating 300 nM (~10-fold Kd app) purified arrestin1 labeled with sulforhodamine at position 348 with increasing concentrations of P-Rh*. The slope of the linear phase corresponds to 0.86 mol/mol, which is close to 0.99 mol/mol we measured previously in native disc membranes with purified arrestin1 in vitro, and to ~0.83:1 we determined in living mice.
Conclusions: :
Arrestin1 binding to monomeric P-Rh* in nanodiscs demonstrates the same affinity and stoichiometry as its binding to P-Rh* in native disc membranes. Thus, monomeric P-Rh* is the physiologically relevant arrestin1 binding partner. This makes perfect sense: biological function of arrestin1 is to quench rhodopsin signaling, and monomeric Rh* was recently found to activate transducin most efficiently.
Keywords: photoreceptors • protein structure/function • signal transduction