Dimerization Domain of Retinal Guanylyl Cyclase (RetGC1) Is an Essential Part of Guanylyl Cyclase Activating Protein (GCAP) Binding Interface [Signal Transduction]

June 22nd, 2015 by Peshenko, I. V., Olshevskaya, E. V., Dizhoor, A. M.

Photoreceptor-specific proteins, GCAPs, bind and regulate retinal membrane guanylyl cyclase, RetGC1, but not natriuretic peptide receptor A, NPRA. Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early-onset blindness disrupted the RetGC1 ability to bind GCAP, but did not eliminate its affinity for another photoreceptor-specific protein - RD3 (retinal degeneration 3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs, but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, yet the region around Met823 was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding, similar to the disease-causing mutation in the neighboring Arg822. The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled-coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high and functional complementation tests showed that RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met823 or Arg822 were mutated. These results directly demonstrate that the interface for GCAPs binding on RetGC1 requires not only kinase homology region, but also directly involves dimerization domain and especially its portion containing Arg822 and Met823.
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Evaluating the Role of Retinal Guanylyl Cyclase 1 (RetGC1) Domains In Binding Guanylyl Cyclase Activating Proteins (GCAP) [Neurobiology]

January 23rd, 2015 by Peshenko, I. V., Olshevskaya, E. V., Dizhoor, A. M.

Retinal membrane guanylyl cyclase 1 (RetGC1) regulated by guanylyl cyclase activating proteins (GCAPs) controls photoreceptor recovery and when mutated causes blinding disorders. We evaluated the principal models of how GCAP1 and GCAP2 bind RetGC1 - through a shared docking interface versus independent binding sites formed by distant portions of the cyclase intracellular domain. At near-saturating concentrations, GCAP1 and GCAP2 activated RetGC1 from HEK293 cells and RetGC2-/-GCAPs1,2-/- mouse retinas in a non-additive fashion. The Met26Arg GCAP1, which binds but does not activate RetGC1, suppressed activation of recombinant and native RetGC1 by competing with both GCAP1 and GCAP2. Untagged GCAP1 displaced both GCAP1-GFP and GCAP2-GFP from the complex with RetGC1 in HEK293 cells. The intracellular segment of a natriuretic peptide receptor A (NPRA) guanylyl cyclase failed to bind GCAPs, but replacing its kinase-homology (KHD) and dimerization (DD) domains with those from RetGC1 restored GCAP1 and GCAP2 binding by the hybrid cyclase and its GCAP-dependent regulation. Deletion of the Tyr1016-Ser1103 fragment in RetGC1 did not block GCAP2 binding to the cyclase. In contrast, substitutions in KHD, Trp708Arg and Ile734Thr, linked to Leber congenital amaurosis (LCA) prevented binding of both GCAP1GFP and GCAP2GFP. Our results demonstrate that GCAPs cannot regulate RetGC1 using independent primary binding sites. Instead, GCAP1 and GCAP2 bind with the cyclase molecule in a mutually exclusive manner using a common or overlapping binding site(s) in the Arg488-Arg851 portion of RetGC1, and mutations in that region causing congenital LCA blindness disrupt activation of the cyclase by both GCAP1 and GCAP2.