Activation of retinal guanylyl cyclase RetGC1 by GCAP1: Stoichiometry of binding and effect of new LCA-related mutations

Academic Article

Abstract

  • Retinal membrane guanylyl cyclase (RetGC) and Ca2+/Mg 2+ sensor proteins (GCAPs) control the recovery of the photoresponse in vertebrate photoreceptors, through their molecular interactions that remain rather poorly understood and controversial. Here we have determined the main RetGC isozyme (RetGC1):GCAP1 binding stoichiometry at saturation in cyto, using fluorescently labeled RetGC1 and GCAP1 coexpressed in HEK293 cells. In a striking manner, the equimolar binding of RetGC1 with GCAP1 in transfected HEK293 cells typical for wild-type RetGC1 was eliminated by a substitution, D639Y, in the kinase homology domain of RetGC1 found in a patient with a severe form of retinal dystrophy, Leber congenital amaurosis (LCA). A similar effect was observed with another LCA-related mutation, R768W, in the same domain of RetGC1. In contrast to the completely suppressed binding and activation of RetGC1 by Mg2+-liganded GCAP1, neither of these two mutations eliminated the GCAP1-independent activity of RetGC stimulated by Mn 2+. These results directly implicate the D639 (and possibly R768)-containing portion of the RetGC1 kinase homology domain in its primary recognition by the Mg2+-bound activator form of GCAP1. © 2010 American Chemical Society.
  • Authors

    Published In

  • Biochemistry  Journal
  • Digital Object Identifier (doi)

    Author List

  • Peshenko IV; Olshevskaya EV; Yao S; Ezzeldin HH; Pittler SJ; Dizhoor AM
  • Start Page

  • 709
  • End Page

  • 717
  • Volume

  • 49
  • Issue

  • 4