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Abstract
The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca2+]. GCAP-1 is a Ca2+-binding protein and belongs to the superfamily of EF-hand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15-amino acid peptide located adjacent to the second EF-hand motif (Phe73-Lys87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe73-Lys87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca2+]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments.
Highlights
Rod and cone photoreceptor cells respond to light by closure of cGMP-gated ion channels in the plasma membrane of the outer segment
We have identified a 15-amino acid region located C-terminally from the second EF-hand (Phe73–Lys87) in guanylyl cyclase-activating protein 1 (GCAP-1) as the main interaction domain (Fig. 3) for rod outer segments (ROS)-GC
The peptide interfered with the guanylyl cyclase-activating protein (GCAP)-1-dependent activation of ROS-GC at low [Ca2ϩ], which indicates that it critically disturbs the Ca2ϩ-dependent activation of ROS-GC by GCAP-1
Summary
Rod and cone photoreceptor cells respond to light by closure of cGMP-gated ion channels in the plasma membrane of the outer segment. Synthesis of cGMP is catalyzed by a membrane-bound retina-specific guanylyl cyclase in rod outer segments (ROS-GC).1 Two isoforms of this enzyme are expressed in photoreceptor cells [5,6,7,8,9,10,11]. The activity of the ROS-GC forms is regulated by a photoreceptor-specific Ca2ϩ-binding protein named guanylyl cyclase-activating protein (GCAP) that is expressed in two isoforms (GCAP-1 and GCAP-2) (18 –22). In the dark, when the [Ca2ϩ ] is high (500 nM), ROS-GC activity is low, and it is stimulated at least 10-fold during the light response, when the [Ca2ϩ] drops to Յ100 nM Both GCAP forms are heterogeneously acylated at the N terminus by C14:0, C14:1, C14:2, and C12:0 fatty acids. Coimmunoprecipitation studies demonstrated the presence of an interacting complex of ROS-GC, GCAP-1, and tubulin independent of [Ca2ϩ]
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