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Ning Li, Izabela Sokal, J. Darin Bronson, Krzysztof Palczewski, Wolfgang Baehr
Identification of Functional Regions of Guanylate Cyclase-Activating Protein 1 (GCAP1) Using GCAP1/GCIP Chimeras
Guanylate cyclaseactivating protein 1 (GCAP1) and guanylate cyclaseinhibitory protein (GCIP) are calmodulinrelated Ca2+binding proteins expressed in vertebrate photoreceptor cells. GCAP1 activates photoreceptor guanylate cyclase 1 (GC1) at low free [Ca2+] (<50 nM, in the light), but inhibits it at physiological high [Ca2+](1 M, in the dark). GCIP, a Ca2+binding protein from frog retina, inhibits GC1 at ~1 M [Ca2+], but is unable to stimulate cyclase at low [Ca2+]. In this study, we probed the interaction between GCAP1 and GC1 by producing GCAP1/GCIP chimeras and tested their capability to stimulate GC1. We prepared eight pairs of constructs in which the Nterminal portions of GCIP and GCAP1 were successively replaced by corresponding domains of GCAP1, and GCIP, respectively. The expressed proteins were purified and tested for stimulation of GC1 at 50 nM [Ca2+], and their ability to competitively inhibit GC1 stimulation by a Ca2+insensitive GCAP1 mutant, GCAP1-tm, at high [Ca2+]. While all GCAP1/GCIP chimeras competitively inhibited GC1 stimulation at high [Ca2+] by GCAP1-tm, several of the GCIP/GCAP1 chimeras had no effect. A chimera consisting of residues 120 of GCIP and 21205 of GCAP1 had no effect on GC1 at low [Ca2+], suggesting that the Nterminal region MGNIMDGKSVEELSSTECHQ, which has no sequence similarity to GCIP, is among the key components necessary for GC1 stimulation. A GCAP1/GCIP chimera consisting of residues 143 (including nonfunctional EF1) of GCAP1 and residues 56206 of GCIP stimulated GC1 at low [Ca2+] and inhibited GC1 at high [Ca2+], suggesting that the essential components required to transform an inhibitory to an activating protein are contained within the Nterminal region of GCAP1 (residues 143).
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 382, 08/2001
Pages: 1179 - 1188