The Calcium-Sensor Guanylate Cyclase Activating Protein Type 2 Specific Site in Rod Outer Segment Membrane Guanylate Cyclase Type 1

Abstract

The rod outer segment membrane guanylate cyclase type 1 (ROS-GC1), originally identified in the photoreceptor outer segments, is a member of the subfamily of Ca(2+)-modulated membrane guanylate cyclases. In phototransduction, its activity is tightly regulated by its two Ca(2+)-sensor protein parts, GCAP1 and GCAP2. This study maps the GCAP2-modulatory site in ROS-GC1 through the use of multiple techniques involving surface plasmon resonance binding studies with soluble ROS-GC1 constructs, coimmunoprecipitation, functional reconstitution experiments with deletion mutants, and peptide competition assays. The findings show that the sequence motif of the core GCAP2-modulatory site is Y965-N981 of ROS-GC1. The site is distinct from the GCAP1-modulatory site. It, however, partially overlaps with the S100B-regulatory site. This indicates that the Y965-N981 motif tightly controls the Ca(2+)-dependent specificity of ROS-GC1. Identification of the site demonstrates an intriguing topographical feature of ROS-GC1. This is that the GCAP2 module transmits the Ca(2+) signals to the catalytic domain from its C-terminal side and the GCAP1 module from the distant N-terminal side.

Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface

“…Contrary to the models hypothesized elsewhere (32)(33)(34), we recently demonstrated that the docking interface(s) for both GCAP1 and GCAP2 is encoded by a region, Arg 488 -Arg 851 , containing the KHD and dimerization domain of RetGC1 (27) rather than its C terminus and transmembrane-proximal portions, making two independent docking sites for different GCAPs (33,35). In the present study, we tested how mutations (site-directed or naturally occurring) specifically in the RetGC1 dimerization domain affected binding of GCAPs.…”

Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic Activity

“…Bicarbonate binds to the CCD. For illustrative purposes, GCAP1, GCAP2, S100B, and neurocalcin ␦ are shown bound to the ROS-GC1 (65)(66)(67)(68)(69). In reality, neurocalcin ␦ is not expressed in photoreceptors, and the ROS-GC1 dimer binds either two GCAP1s or two GCAP2s.…”

Evaluating the Role of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Domains in Binding Guanylyl Cyclase-activating Proteins (GCAPs)

“…The mOrangeRetGC1VenusN chi- Fig. 2; the a max values for stimulation by GCAPs in the absence or presence of 1 and 2 M M26R GCAP1 only slightly change for stimulation by GCAP1 (27,26, and 25 nmol of cGMP/min/mg, respectively) or GCAP2 (22,21, and 19 nmol of cGMP/min/mg, respectively), but the K 1/2GCAP increased in the presence of M26R GCAP1 for wild type GCAP1 (1.5, 4, and 6.7 M, respectively) and GCAP2 (1.7, 4.7, and 7.4 M, respectively). The Hill coefficient increased for GCAP1 (from 1.17 in the absence to 1.53 in the presence of 2 M M26R GCAP1) and for GCAP2 (from 1.14 in the absence to 1.54 in the presence of 2 M M26R GCAP1).…”

“…6, top), and thus eliminated the entire putative Tyr 1016 -Ser 1103 GCAP2 binding site (26,27), with a non-homologous sequence: the N-terminal portion of the YFP, VenusN (49). The chimera protein lacking the putative GCAP2 binding site was then tested for co-localization with GCAP2 in a cell-based binding assay (Fig.…”

“…Not only do NPRA and RetGC1 share little homology between their C-terminal portions, but the Tyr 1016 -Ser 1103 region in RetGC1 hypothesized to contain the GCAP2 binding site (26,27) is one of the least homologous regions between the two cyclases (2), and most of it is merely absent from NPRA (Fig. 9, top).…”