June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Point mutations in guanylyl cyclase RetGC1 dimerization domain disrupt binding of GCAP1 and GCAP2.
Author Affiliations & Notes
  • Igor V Peshenko
    Salus University, Elkins Park, PA
  • Elena V Olshevskaya
    Salus University, Elkins Park, PA
  • Alexander M Dizhoor
    Salus University, Elkins Park, PA
  • Footnotes
    Commercial Relationships Igor Peshenko, None; Elena Olshevskaya, None; Alexander Dizhoor, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4654. doi:
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      Igor V Peshenko, Elena V Olshevskaya, Alexander M Dizhoor; Point mutations in guanylyl cyclase RetGC1 dimerization domain disrupt binding of GCAP1 and GCAP2.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4654.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: Guanylyl cyclase activating proteins (GCAPs) bind photoreceptor guanylyl cyclase 1 (RetGC1) and regulate its activity in a Ca2+-sensitive manner. The motifs in RetGC1 primary structure forming GCAP binding interface are poorly understood and the role of RetGC domain structure in forming the complex with GCAPs remains controversial. We tested a potential role of the cyclase dimerization domain (DD) in its interaction with GCAPs.

Methods: Multiple mutated forms of RetGC1 tagged with mOrange fluorescent protein were co-expressed with GCAP1-GFP and GCAP2-GFP in HEK293 cells to test their co-localization [1]. Mutants incapable of binding GCAPs were also functionally tested for their ability to complement RetGC1AC mutant, in which the active site was converted into that of adenylyl cyclase [2].

Results: Replacement of the DD domain in RetGC1 with that of a natriuretic peptide receptor cyclase (NPRA) renders RetGC1 incapable of binding both GCAP1 and GCAP2. Shuffling the sequences between NPRA and RetGC1 in the DD domain revealed that the specificity of RetGC1 DD domain to enable GCAP binding critically depended on a single residue, Met823 - replacement of Met823 with Arg from NPRA blocked binding of both GCAP1 and GCAP2 and inactivated the cyclase activation by either GCAP. At the same time, computation argues that the M823R could not decrease the ability of the DD domain to form a coiled-coil structure in a catalytically active cyclase dimer. To verify that the mutated RetGC1 was able to form such a dimer, we co-expressed it with RetGC1AC(-) mutant, whose active site was converted from GTP binding to ATP binding [2] but also lacked a side chain essential for the catalysis. The M823R RetGC1 restored adenylyl cyclase activity of the RetGC1AC(-) by positioning the missing residue to the active site formed by the heterodimer while the RetGC1AC(-) enabled GCAP-dependent activation of the complex. We also found that an LCA-linked neighboring mutation, R822P, blocked GCAPs binding despite not being a part of the coiled-coil interface.

Conclusions: We conclude that dimerization domain, in addition to its participation in cyclase dimer formation, presents one of the essential elements of a complex GCAP binding interface in the RetGC1 primary structure. Reference. [1] Peshenko et al.(2008) . J Biol Chem. 283, 21747-21757; [2] Tucker et al. (1998) Proc Natl Acad Sci U S A 95, 5993-5997


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