Abstract
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.
Highlights
Guanylyl cyclase activating proteins (GCAPs)2 are Ca2ϩ/ Mg2ϩ-binding proteins that regulate retinal guanylyl cyclase (RetGC), the enzyme that supplies photoreceptors with the phototransduction messenger, cGMP [1,2,3,4]
These results directly demonstrate that metal binding in EF-hand 2 is crucial for Guanylyl cyclase activating protein 1 (GCAP1) attachment to RetGC1, and that in EF-hand 3 it is less critical, it enhances the efficiency of the GCAP1 docking on the target enzyme
Following RetGC1—It was important for the entire line of the study to elctroblotting on Immobilon P membrane (Millipore), proteins establish whether or not the fluorescent tags can affect the were probed with the rabbit polyclonal anti-RetGC1 and anti- activity of GCAP1 or RetGC1
Summary
Recombinant dsRed-tagged RetGC1—Recombinant RetGC1 was expressed in HEK293 cells from a modified pRCCMV vector (Stratagene), as previously described [24]. Labeled RetGC1 was produced by inserting in RetGC1 cDNA a DNA fragment coding for a monomeric red fluorescent protein, dsRed (Clontech), PCR-amplified with the BstEII/KpnI restriction sites at the ends. To express RetGC1 for the functional assay in vitro, HEK293 cells were transfected with 40 g/100-mm culture dish of pRCCMV plasmid containing wild type RetGC1 or dsRed-RetGC1 using Ca2ϩ-phosphate method (a Promega Profection protocol), and the membranes were harvested as previously described [24]. Co-expression of RetGC1 and GCAP1 in HEK293 Cells and Confocal Laser Scanning Microscopy—Cells were grown in standard glass coverslip chambers (four 2-cm chambers per slide) in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, and were transfected with a mixture of expression constructs using the Ca2ϩ-phosphate method. The same software was used for a non-paired t test to compare the data for the D64N mutant versus the wild GCAP1-GFP type expressed in the absence of the cyclase
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