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Vsevolod V Gurevich, Mira Bella May, Preethi Karnam, Chen Zheng, Sergey A Vishnivetskiy; The mechanism of phosphate sensing by arrestin-1. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1523.
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© ARVO (1962-2015); The Authors (2016-present)
Determine the role of conserved lysine in arrestin lariat loop in phosphate recognition.
Mutations eliminating or reversing the charge of Lys-300 in bovine and homologous Lys-301 in mouse arrestin-1 were introduced on the background of wild type and several “enhanced” phosphorylation-independent mutants of these proteins. Functional effects of these mutations were tested in direct binding assay using radiolabeled proteins produced in cell-free translation.
Conserved lysine between two negative charges in the lariat loop that participate in the polar core in all arrestin subtypes directly binds receptor- or IP6-attached phosphates in active arrestin crystal structures. This suggested that receptor-attached phosphate might activate arrestins by “pulling” the lariat loop out of the polar core via this lysine. In this case elimination and reversal of its charge would be expected to greatly reduce arrestin-1 binding to active phosphorylated rhodopsin (P-Rh*), but have minimal effect of the binding of enhanced mutants to active unphosphorylated rhodopsin (Rh*). We found that Lys->Ala mutation in all cases yields less than 20% reduction in P-Rh* binding, and even Lys->Glu mutation does not reduce P-Rh* binding more than 40%, which is not consistent in a key role of this lysine in phosphate-dependent arrestin activation. Moreover, we found that in the context of enhanced mutants these mutations significantly affect Rh* binding, decreasing or enhancing it in different cases. The direction and magnitude of the effect of Lys->Ala and Lys->Glu mutations on Rh* binding depends on the context. In every case the effect of these substitutions on Rh* binding was greater than their effect on P-Rh* binding.
Conserved lysine between two negative charges in the lariat loop participates in rhodopsin binding, but it plays much greater role in the interaction with non-phosphorylated parts of the receptor than with receptor-attached phosphates.
This is a 2020 ARVO Annual Meeting abstract.
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