Binding of guanylyl cyclase activating protein 1 (GCAP1) to retinal guanylyl cyclase (RetGC1). The role of individual EF-hands.
Guanylyl cyclase activating protein 1 (GCAP1), after substitution of Ca(2+) by Mg(2+) in its EF-hands, stimulates photoreceptor guanylyl cyclase, RetGC1, in response to light. We inactivated metal binding in individual EF-hands of GCAP1 tagged with green fluorescent protein to assess their role in GCAP1 binding to RetGC1 in co-transfected HEK293 cells. When expressed alone, GCAP1 was uniformly distributed throughout the cytoplasm and the nuclei of the cells, but when co-expressed with either fluorescently tagged or non-tagged RetGC1, it co-localized with the cyclase in the membranes. The co-localization did not occur when the C-terminal portion of RetGC1, containing its regulatory and catalytic domains, was removed. Mutations that preserved Mg(2+) binding in all three metal-binding EF-hands did not affect GCAP1 association with the cyclase in live cells. Locking EF-hand 4 in its apo-conformation, incapable of binding either Ca(2+) or Mg(2+), had no effect on GCAP1 association with the cyclase. In contrast to EF-hand 4, inactivation of EF-hand 3 reduced the efficiency of the co-localization, and inactivation of EF-hand 2 drastically suppressed GCAP1 binding to the cyclase. These results directly demonstrate that metal binding in EF-hand 2 is crucial for GCAP1 attachment to RetGC1, and that in EF-hand 3 it is less critical, although it enhances the efficiency of the GCAP1 docking on the target enzyme. Metal binding in EF-hand 4 has no role in the primary attachment of GCAP1 to the cyclase, and it only triggers the activator-to-inhibitor functional switch in GCAP1.
Hafter Research Laboratories, Pennsylvania College of Optometry, 8360 Old York Road, Elkins Park, PA 19027, USA.
Igor V
IV
Peshenko
Elena V
EV
Olshevskaya
Alexander M
AM
Dizhoor
D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D018613:Microscopy, Confocal; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012160:Retina / Q000201:enzymology*
D051604:Guanylate Cyclase-Activating Proteins; D011994:Recombinant Proteins; D006162:Guanylate Cyclase; D008274:Magnesium