Ca2+–Modulated GCAP2 Site in the Phototransduction–Linked Rod Outer Segment Membrane Guanylate Cyclase, ROS–GC1

Abstract

Abstract: : Purpose:ROS–GC1 is a photoreceptor membrane guanylate cyclase, which through two Ca²⁺ sensor proteins, GCAP1 and GCAP2, is linked with phototransduction. GCAP1 and GCAP2 modulated domains in ROS–GC1 are distinct. In this investigation the GCAP2–modulated site has been mapped. Methods: Mutagenesis/expression/reconstitution experiments, peptide competition, real–time binding (surface plasmon resonance spectroscopy) experiments, co–immunoprecipitation and cross–linking studies were carried out. Results:Co–immunoprecipitation and SPR experiments show that the ROS–GC1 soluble fragment aa 731–1054 binds GCAP2 (KD value of 3.7 µM as determined by SPR) but does not bind GCAP1. The ROS–GC1 truncated mutant lacking aa 965–1054 loses GCAP2–dependent activity whereas its behavior toward GCAP1 remains unaffected. Direct binding of GCAP2 to the ROS–GC1 fragment aa 965–1054 is evidenced by co–immunoprecipitation and SPR. The binding occurs with and without Ca²⁺ with KD value of about 1.7 µM. Peptide competition experiments indicate that there is one GCAP2 binding site in ROS–GC1. Conclusions:The ROS–GC1–GCAP2 signaling pathway operates via a specific site on ROS–GC1; the site is distinct from the GCAP1–modulatory site, and GCAP2 binding to ROS–GC1 is Ca²⁺–independent.

Keywords: signal transduction • second messengers • calcium