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.

Exchange protein directly activated by cAMP-1 (Epac1) is a cAMP sensor that regulates multiple cellular functions including cellular migration, proliferation and differentiation. Classically, Epac1 is thought to exert its effects through binding of cAMP leading to a conformational change in Epac1 and its accumulation at the plasma membrane (PM) where it activates Rap1. In search for regulators of Epac1 activity, we show here that importin β1 (impβ1) is an Epac1 binding partner that prevents PM accumulation of Epac1. We demonstrate that in the absence of impβ1, endogenous as well as overexpressed Epac1 accumulate at the PM. Moreover, agonist-induced PM translocation of Epac1 leads to dissociation of Epac1 from impβ1. Localization of Epac1 at the PM in the absence of impβ1, requires residue R82 in its DEP domain. Notably, the PM accumulation of Epac1 in the absence of impβ1 does not require binding of cAMP to Epac1 and does not result in Rap1 activation. Functionally, PM accumulation of Epac1, an Epac1 mutant deficient in cAMP binding, or an Epac1 mutant tethered to the PM, is sufficient to inhibit neurite outgrowth. In conclusion, we uncover a cAMP-independent function of Epac1 at the PM and demonstrate that impβ1 controls subcellular localization of Epac1.

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization.