A Functional Kinase Homology Domain Is Essential for the Activity of Photoreceptor Guanylate Cyclase 1

Grzegorz Bereta,Benlian Wang,Philip D Kiser,Wolfgang Baehr,Geeng-Fu Jang,Krzysztof Palczewski

doi:10.1074/jbc.m109.061713

Abstract

Phototransduction is carried out by a signaling pathway that links photoactivation of visual pigments in retinal photoreceptor cells to a change in their membrane potential. Upon photoactivation, the second messenger of phototransduction, cyclic GMP, is rapidly degraded and must be replenished during the recovery phase of phototransduction by photoreceptor guanylate cyclases (GCs) GC1 (or GC-E) and GC2 (or GC-F) to maintain vision. Here, we present data that address the role of the GC kinase homology (KH) domain in cyclic GMP production by GC1, the major cyclase in photoreceptors. First, experiments were done to test which GC1 residues undergo phosphorylation and whether such phosphorylation affects cyclase activity. Using mass spectrometry, we showed that GC1 residues Ser-530, Ser-532, Ser-533, and Ser-538, located within the KH domain, undergo light- and signal transduction-independent phosphorylation in vivo. Mutations in the putative Mg(2+) binding site of the KH domain abolished phosphorylation, indicating that GC1 undergoes autophosphorylation. The dramatically reduced GC activity of these mutants suggests that a functional KH domain is essential for cyclic GMP production. However, evidence is presented that autophosphorylation does not regulate GC1 activity, in contrast to phosphorylation of other members of this cyclase family.

Highlights

In photoreceptor outer segments, photoreceptor guanylate cyclases GC1 and GC23 produce cGMP, the second messenger of phototransduction [1,2,3,4]
We demonstrate that mutations in the putative Mg2ϩ binding site within the kinase homology (KH) domain located in the primary sequence far away from the phosphorylation site are crucial for GC1 and protein kinase activities, suggesting regulation of cGMP production and autophosphorylation by the KH domain
Identification of the Phosphorylated Residues in Mouse GC1— Initially, to determine which region of GC1 undergoes phosphorylation, we performed mass spectrometry analysis of GC1 purified from bovine rod outer segments (ROS) and found a doubly phosphorylated VAQGpSRTpSLAAR peptide from the N-terminal region of the KH domain

Summary

Introduction

Photoreceptor guanylate cyclases GC1 and GC2 ( known as GC-E and GC-F) produce cGMP, the second messenger of phototransduction [1,2,3,4] (reviewed in Refs. 5 and 6). Photoreceptor GCs belong to a family of membrane-bound GCs composed of an extracellular (EC), transmembrane (TM), kinase homology (KH), dimerization (DIM), and catalytic (CAT) domain (Fig. 1A) How these domains cooperate to achieve precisely regulated cGMP synthesis was proposed for a homolog of GC1, the natriuretic peptide receptor A (NPR-A). The KH domain of GC1 might undergo autophosphorylation, a surprising finding considering that the invariable “catalytic” Asp residue required for efficient kinase activity is absent in this enzyme [26] The function of this phosphorylation is unknown. We demonstrate that mutations in the putative Mg2ϩ binding site within the KH domain located in the primary sequence far away from the phosphorylation site are crucial for GC1 and protein kinase activities, suggesting regulation of cGMP production and autophosphorylation by the KH domain. Our data from studies of wild-type (WT) and knock-out mice provide evidence that phosphorylation of photoreceptor GCs is independent of GCAPs and either direct or indirect activation by light

Methods

Results

Conclusion