Abstract
Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin alpha-subunit were mostly unaffected. Outer segment membranes of GC1-/- and GC double knock-out cones were destabilized and devoid of cone transducin (alpha- and gamma-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the down-regulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated phototransduction proteins.
Highlights
MARCH 23, 2007 VOLUME 282 NUMBER 12 brane proteins with a single transmembrane domain [1,2,3]
Generation of GC2Ϫ/Ϫ and GC1/GC2 Double Knock-out (GCdko) Mice—The GC1 (Gucy2e, on mouse chromosome 11) and the GC2 (Gucy2f, on the X chromosome) genes are closely related in structure (Fig. 1, A and B) [34]
Immunoblots of GC1Ϫ/Ϫ and GC2Ϫ/Ϫ retinal lysates probed with monoclonal anti-GC1 and polyclonal anti-GC2 antibodies confirmed that GC1 and GC2 were not expressed (Fig. 1E)
Summary
MARCH 23, 2007 VOLUME 282 NUMBER 12 brane proteins with a single transmembrane domain [1,2,3]. GC1 is detected in the retina, pineal gland, and olfactory bulb [4] as well as the cochlear nerve and the organ of Corti [5], whereas GC2 is found only in the retina The activities of these enzymes are Ca2ϩ-sensitive, a sensitivity that is mediated by guanylate cyclase-activating proteins (GCAPs). Of the two GCAPs present in mouse retina, GCAP1 stimulates GC1 more efficiently than GC2 [11] Deletion of both GCAP genes delays recovery of the dark current due to loss of Ca2ϩ-dependent GC activation [12]. Deletion of GC1 and GC2 in Retinal Photoreceptors tion of PDE6 and GCAPs in GCdko rods as well as down-regulation of cone phototransduction components in GC1Ϫ/Ϫ cones suggest that GCs serve enzymatic, stabilizing, and structural/transport roles in photoreceptors
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