Lysine in the lariat loop of arrestins does not serve as phosphate sensor

Abstract

Protein-protein interactions govern virtually all essential cellular functions. In most cases, proteins bind only a certain functional state of their partners, while "ignoring" other states. Thus, elucidation of molecular mechanisms whereby one protein "selects" a particular functional state of its binding partner has general biological significance. Arrestins are among the most studied proteins in this regard. Arrestins selectively interact with phosphorylated and activated forms of their cognate G-protein-coupled receptors (GPCRs), binding them with much higher affinity than the same receptors when they are inactive or unphosphorylated (reviewed in (Carman & Benovic, 1998; Gurevich & Gurevich, 2004)). Vertebrates express four arrestin isoforms: two specialized visual in photoreceptors, arrestin-1 1 and-4, and two ubiqui-1 We use systematic names of arrestin proteins, where the number after the dash indicates the order of cloning: arrestin-1 (historic names S-antigen, 48 kDa protein, visual or rod arrestin), arrestin-2 (β-arrestin or β-arrestin1), arrestin-3 (β-arrestin2 or hTHY-ARRX), and arrestin-4 (cone or X-arrestin).

Receptor-Arrestin Interactions: The GPCR Perspective

“…Most importantly, simultaneous charge reversal of both residues, restoring the salt bridge, also restored wild-type (WT) arrestin-1 selectivity for P-Rh* [16,28]. Charge reversal of homologous arginines in non-visual arrestin-2 and -3 also greatly enhanced the binding to unphosphorylated forms of their cognate receptors [23,24,[29][30][31], as could be expected if this phosphate-sensing mechanism is shared by all members of the arrestin family. Thus, the issue appeared to be settled: R175 and its homologues in other subtypes act as phosphate sensors-phosphate binding neutralizes the charge of the arginine, breaking the salt bridge with the homologue of D296, which destabilizes the polar core, thereby "telling" arrestin to swing into action (reviewed in [2]).…”

“…This model did not survive experimental testing either. Elimination or even reversal of the charge of this lysine in bovine and mouse arrestin-1, as well as in arrestin-2 and -3, did not dramatically reduce their binding to the cognate receptors, as this model predicted [31].…”

“…The lariat loop (N281-N311 in arrestin-2, dark blue in Figure 1B), which is highly conserved in all arrestins, contains the main counterion of R169 in the polar core, D290. Mutagenesis data suggest that the salt bridge between these two residues plays a central role in stabilizing the basal state [23,24,[28][29][30][31]. The gate loop [16] (part of the lariat loop, D290-N299 in arrestin-2; dark red in Figure 1B) supplies two out of the three negative charges in the polar core.…”

Biochemical insights into structure and function of arrestins

“…However, this hypothesis was subsequently discarded. Vishnivetskiy et al [67] tested binding of K300A-arr-1 (neutralized mutant) and K300E-arr-1 (charge-inverted mutant) to Rho*-P. They could not confirm a critical role of K300 as a phosphate sensor. Instead, mutagenesis of K300 significantly affected arr-1 binding to nonphosphorylated Rho, suggesting that this residue interacts with nonphosphorylated receptor parts.…”

“…Vishnivetskiy et al . [67] tested binding of K300A‐arr‐1 (neutralized mutant) and K300E‐arr‐1 (charge‐inverted mutant) to Rho*‐P. They could not confirm a critical role of K300 as a phosphate sensor.…”