The kinase homology domain of retinal guanylyl cyclases 1 and 2 specifies the affinity and cooperativity of interaction with guanylyl cyclase activating protein-2.
RetGC-1 and RetGC-2 are photoreceptor membrane guanylyl cyclases that are regulated by the Ca2+-binding protein, GCAP-2. We used a protease protection assay to localize regions of the intracellular domains of RetGCs important for the interaction with GCAP-2 and found that GCAP-2 reduces the access of trypsin to a site in the kinase homology domain (KHD) of RetGC-1. The protective effect of GCAP-2 is independent of Ca2+. We also found that RetGC-2 and GCAP-2 interact cooperatively with high affinity, but RetGC-1 and GCAP-2 interact noncooperatively with low affinity. By analyzing RetGC-1/RetGC-2 chimeras we demonstrated that the affinity and cooperativity of the interaction with GCAP-2 is dictated by the structure of the KHD. These findings suggest that GCAP-2 interacts constituitively with the KHDs of RetGC-1 and RetGC-2 and that cGMP synthesis is controlled by Ca2+-dependent conformational changes in the RetGC/GCAP complex.
Department of Biochemistry, Howard Hughes Medical Institute, University of Washington, Seattle 98195-7370, USA.
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D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D004533:Egtazic Acid / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012160:Retina / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D002135:Calcium-Binding Proteins; C496736:GUCA1A protein, human; C496739:GUCA1B protein, human; D051604:Guanylate Cyclase-Activating Proteins; D011993:Recombinant Fusion Proteins; D004533:Egtazic Acid; D010770:Phosphotransferases; D010450:Endopeptidases; D006162:Guanylate Cyclase