C-terminal threonines and serines play distinct roles in the desensitization of rhodopsin, a G protein-coupled receptor

Abstract

Rod photoreceptors generate measurable responses to single-photon activation of individual molecules of the G protein-coupled receptor (GPCR), rhodopsin. Timely rhodopsin desensitization depends on phosphorylation and arrestin binding, which quenches G protein activation. Rhodopsin phosphorylation has been measured biochemically at C-terminal serine residues, suggesting that these residues are critical for producing fast, low-noise responses. The role of native threonine residues is unclear. We compared single-photon responses from rhodopsin lacking native serine or threonine phosphorylation sites. Contrary to expectation, serine-only rhodopsin generated prolonged step-like single-photon responses that terminated abruptly and randomly, whereas threonine-only rhodopsin generated responses that were only modestly slower than normal. We show that the step-like responses of serine-only rhodopsin reflect slow and stochastic arrestin binding. Thus, threonine sites play a privileged role in promoting timely arrestin binding and rhodopsin desensitization. Similar coordination of phosphorylation and arrestin binding may more generally permit tight control of the duration of GPCR activity.DOI: http://dx.doi.org/10.7554/eLife.05981.001

Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors

“…Indeed, similar rhodopsin mutants with single or multiple serine/threonine residues substituted with alanine have been reported to prolong reproducible single-photon response, i.e. rhodopsin deactivation and recovery (Azevedo et al, 2015; Mendez et al, 2000). We further tested the effects of the phosphorylation code of V2R C-tail on arrestin binding by mutations of S357A/T360A/S363A (3A) or S357A/T360A/S362A/S363A/S364A (6A) that respectively disrupt either the first phosphorylation code SCTTAS or both codes SCTTAS and SCTTASS on the V2R C-tail of a β 2 AR-V2R chimeric receptor.…”

How rods respond to single photons: Key adaptations of a G‐protein cascade that enable vision at the physical limit of perception

“…Although arrestin binds poorly to singly phosphorylated Rh*, it can bind almost fully when already three of the six sites are phosphorylated . Sites are probably not phosphorylated at random, because Kennedy and coworkers have shown that serines are among the first to be phosphorylated , whereas the recent results of Azevedo and coworkers indicate that phosphorylation of threonines is especially important for terminating Rh* activity. Despite clever and determined attempts by many investigators, we still do not know the specificity of the kinase or time course of its action.…”

Spatial and Temporal Aspects of Signaling by G-Protein–Coupled Receptors

“…We have seen above how rhodopsin, by its funnel-like interaction process, can manage to interact with its signaling proteins both fast and precisely. The catalytic process lasts until R* becomes deactivated, generating a surprisingly uniform single quantum response (see Azevedo et al, 2015). Current estimates of R* lifetime in mouse rods are in the range of ∼40 milliseconds (Gross and Burns, 2010).…”