Mapping functional domains of the guanylate cyclase regulator protein, GCAP-2.
Guanylate cyclase regulator protein (GCAP)-2 is a Ca2+-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca2+-sensitive manner. GCAP-2 activates RetGC at free Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca2+ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca2+-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe78 and Asp113 determines whether GCAP-2 activates outer segment RetGC in low or high Ca2+ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca2+ concentrations. 2) The amino acid sequence of GCAP-2 between Lys29 and Phe48 that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca2+ and inhibition at high Ca2+ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val171-Asn189, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca2+-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.
Olshevskaya
E V
EV
Department of Ophthalmology/Kresge Eye Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Boikov
S
S
Ermilov
A
A
Krylov
D
D
Hurley
J B
JB
Dizhoor
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D051604:Guanylate Cyclase-Activating Proteins; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010786:Photoreceptor Cells / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002135:Calcium-Binding Proteins; C496739:GUCA1B protein, human; D051604:Guanylate Cyclase-Activating Proteins; D011994:Recombinant Proteins