Guanylate‐cyclase‐inhibitory protein is a frog retinal Ca²⁺‐binding protein related to mammalian guanylate‐cyclase‐activating proteins

Abstract

Two guanylate-cyclase-activating proteins (GCAP) encoded by a tail-to-tail gene array have been characterized in the mammalian retina. Using frog retina as a model, we obtained evidence for the presence of a photoreceptor Ca 2ϩ -binding protein closely related to GCAP. This protein (206 amino acids) does not stimulate guanylate cyclase (GC) in low [Ca 2ϩ ], but inhibits GC in high [Ca 2ϩ ], and is therefore termed guanylate-cyclase-inhibitory protein (GCIP). Sequence analysis indicates that GCIP and GCAP1 and GCAP2 have diverged substantially, but conserved domains present in all vertebrate GCAP are present in GCIP. Moreover, partial characterization of the GCIP gene showed that the positions of two introns in the GCIP gene are identical to positions of corresponding introns of the mammalian GCAP gene array. As to the major differences between GCIP and GCAP, the fourth EF hand Ca 2ϩ -binding motif of GCIP is disabled for Ca 2ϩ binding, and GCIP does not stimulate GC. Monoclonal and polyclonal antibodies raised against recombinant GCIP identified high levels of GCIP in the inner segments, somata and synaptic terminals of frog cone photoreceptors. The results suggest that GCIP is a Ca 2ϩ -binding protein of the GCAP/recoverin subfamily. Its localization in frog cones closely resembles that of GC in mammalian cones. GCIP inhibits GC at high free [Ca 2ϩ ], competing with GCAP1 and GCAP2 for GC regulatory sites.

Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors

“…3A). These results suggest that GCAP1 and GCAP2 entirely account for Ca 2ϩ regulation of GC in mouse retinas, and that other recently characterized GC-regulatory proteins, such as GCIP in frog (27) and GCAP3 in higher vertebrates (10), are not expressed or cannot compensate for the absence of GCAP1 and GCAP2 in mouse photoreceptors.…”

Activation of Retinal Guanylyl Cyclase-1 by Ca2+-binding Proteins Involves Its Dimerization

“…1 and 3-7). These observations exclude the possibility that inhibitory regulators, such as guanylyl cyclaseinhibitory protein (40) and RGS9 (41), are complexed with retGC-1 to form the 210-kDa product. Using a RGS9-specific antibody (41) we have shown that RGS9 is not involved in the 210-kDa product (Fig.…”

“…Therefore, it is believed that S100 proteins are not involved in phototransduction. Additional mechanisms and factors may also be involved in the regulation of retGCs, including phosphorylation (34,35), ATP binding (36 -38), actin binding (39), an inhibitor (guanylyl cyclase-inhibitory protein) of GCAP-activated retGC in amphibian retina (40), and inhibition of retGC-1 by RGS9 (41).…”

A Possible Role of RGS9 in Phototransduction

“…We note that the inhibitory activity was coeluted with GCBP-55 from both the immunoaffinity and the ion exchange (Econo S) columns. GCBP-55 is not the GC inhibitor reported recently (14), because amino acid sequences in these proteins are completely different, as described below.…”

“…Activation of GC by Ca 2ϩ /GCAPs has been established (11-13); however, a protein inhibitor on GC activity has also been isolated from amphibian photoreceptors (14). Moreover, GC isoforms (10,(15)(16)(17)(18) as well as GC phosphorylation (19,20) have been described, although the physiological significance of this GC diversity is unknown.…”