Dimerization of guanylyl cyclase-activating protein and a mechanism of photoreceptor guanylyl cyclase activation.
Ca(2+)-binding guanylyl cyclase-activating proteins (GCAPs) stimulate photoreceptor membrane guanylyl cyclase (retGC) in the light when the free Ca(2+) concentrations in photoreceptors decrease from 600 to 50 nM. RetGC activated by GCAPs exhibits tight dimerization revealed by chemical cross-linking (Yu, H., Olshevskaya, E., Duda, T., Seno, K., Hayashi, F., Sharma, R. K., Dizhoor, A. M., and Yamazaki, A. (1999) J. Biol. Chem. 274, 15547-15555). We have found that the Ca(2+)-loaded GCAP-2 monomer undergoes reversible dimerization upon dissociation of Ca(2+). The ability of GCAP-2 and its several mutants to activate retGC in vitro correlates with their ability to dimerize at low free Ca(2+) concentrations. A constitutively active GCAP-2 mutant E80Q/E116Q/D158N that stimulates retGC regardless of the free Ca(2+) concentrations forms dimers both in the absence and in the presence of Ca(2+). Several GCAP-2/neurocalcin chimera proteins that cannot efficiently activate retGC in low Ca(2+) concentrations are also unable to dimerize in the absence of Ca(2+). Additional mutation that restores normal activity of the GCAP-2 chimera mutant also restores its ability to dimerize in the absence of Ca(2+). These results suggest that dimerization of GCAP-2 can be a part of the mechanism by which GCAP-2 regulates the photoreceptor guanylyl cyclase. The Ca(2+)-free GCAP-1 is also capable of dimerization in the absence of Ca(2+), but unlike GCAP-2, dimerization of GCAP-1 is resistant to the presence of Ca(2+).
Olshevskaya
E V
EV
Department of Ophthalmology/Kresge Eye Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Ermilov
A N
AN
Dizhoor
A M
AM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004789:Enzyme Activation; D004926:Escherichia coli; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; D011993:Recombinant Fusion Proteins; D006162:Guanylate Cyclase; D002118:Calcium