title: Identification of functional regions of guanylate cyclase-activating protein 1 (GCAP1) using GCAP1/GCIP chimeras.
abstract: Guanylate cyclase-activating protein 1 (GCAP1) and guanylate cyclase-inhibitory protein (GCIP) are calmodulin-related 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 microM, in the dark). GCIP, a Ca2+-binding protein from frog retina, inhibits GC1 at approximately 1 microM [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 N-terminal 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 1-20 of GCIP and 21-205 of GCAP1 had no effect on GC1 at low [Ca2+], suggesting that the N-terminal 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 1-43 (including nonfunctional EF1) of GCAP1 and residues 56-206 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 N-terminal region of GCAP1 (residues 1-43).
authors.affiliation: Moran Eye Center, University of Utah, Salt Lake City 84132, USA.
authors.forename: N
authors.initials: N
authors.lastname: Li
authors.forename: I
authors.initials: I
authors.lastname: Sokal
authors.forename: J D
authors.initials: JD
authors.lastname: Bronson
authors.forename: K
authors.initials: K
authors.lastname: Palczewski
authors.forename: W
authors.initials: W
authors.lastname: Baehr
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins; D007313:Insecta / Q000166:cytology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D029867:Xenopus Proteins
chemical_list: C485815:CCNDBP1 protein, human; D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D051604:Guanylate Cyclase-Activating Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014157:Transcription Factors; D029867:Xenopus Proteins; C110715:guanylate cyclase 1; C450444:guanylate cyclase-inhibitory protein, Xenopus; D006162:Guanylate Cyclase; D002118:Calcium