Lysine in the lariat loop of arrestins does not serve as phosphate sensor. Issue 4 (11th July 2020)

• Record Type: Journal Article
• Authors: Vishnivetskiy, Sergey A.; Zheng, Chen; May, Mira B.; Karnam, Preethi C.; Gurevich, Eugenia V.; Gurevich, Vsevolod V.
• Is Part Of: Journal of neurochemistry. Volume 156:Issue 4(2021)
• Page Start: 435
• Page End: 444
• Publication Date: 2020-07-11
• Subjects: arrestin -- GPCR -- protein–protein interactions -- receptor‐attached phosphates -- structure‐function; Neurochemistry -- Periodicals; 616.8042
• DOI: 10.1111/jnc.15110
• Languages: English
• ISSNs: 0022-3042

Abstract

Abstract: Arrestins demonstrate strong preference for phosphorylated over unphosphorylated receptors, but how arrestins "sense" receptor phosphorylation is unclear. A conserved lysine in the lariat loop of arrestins directly binds the phosphate in crystal structures of activated arrestin‐1, ‐2, and ‐3. The lariat loop supplies two negative charges to the central polar core, which must be disrupted for arrestin activation and high‐affinity receptor binding. Therefore, we hypothesized that receptor‐attached phosphates pull the lariat loop via this lysine, thus removing the negative charges and destabilizing the polar core. We tested the role of this lysine by introducing charge elimination (Lys‐>Ala) and reversal (Lys‐>Glu) mutations in arrestin‐1, ‐2, and ‐3. These mutations in arrestin‐1 only moderately reduced phospho‐rhodopsin binding and had no detectable effect on arrestin‐2 and ‐3 binding to cognate non‐visual receptors in cells. The mutations of Lys300 in bovine and homologous Lys301 in mouse arrestin‐1 on the background of pre‐activated mutants had variable effects on the binding to light‐activated phosphorylated rhodopsin, while affecting the binding to unphosphorylated rhodopsin to a greater extent. Thus, conserved lysine in the lariat loop participates in receptor binding, but does not play a critical role in phosphate‐induced arrestin activation. Abstract : Conserved lysine in the lariat loop of arrestins (K301 in mouse arrestin‐1; PDB 5W0P) contacts receptor‐attached phosphates in crystal structures. Two polar core negative charges are on the same lariat loop. Previous studies showed that destabilization of the polar core by receptor‐attached phosphates is necessary for arrestin activation. Thus, the structures suggest that this lysine might serve as the phosphate sensor of arrestins. We tested this hypothesis by mutagenesis in mouse and bovine arrestin‐1, and in non‐visual arrestins. The data show that lariat loop lysine is not a phosphate sensor, but participates in arrestin interactions with non‐phosphorylated receptor elements.