Iswari Subbaraya

Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase-activating protein

Guanylyl cyclase-activating protein (GCAP) is thought to mediate Ca(2+)-sensitive regulation of guanylyl cyclase (GC), a key event in recovery of the dark state of rod photoreceptors following light exposure. Here, we characterize GCAP from several vertebrate species by molecular cloning and provide evidence that GCAP contains a heterogeneously acylated N-terminal region that interacts with GC. Vertebrate GCAPs consist of 201-205 amino acids, and sequence analysis indicates the presence fo three EF hand Ca(2+)-binding motifs. These results establish that GCAP is a novel photoreceptor-specific member of a large family of Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-sensitive activation of GC.

Guanylyl cyclase activating protein: A calcium-sensitive regulator of phototransduction

Guanylyl cyclase activating protein (GCAP1) has been proposed to act as a calcium-dependent regulator of retinal photoreceptor guanylyl cyclase (GC) activity. Using immunocytochemical and biochemical methods, we show here that GCAP1 is present in rod and cone photoreceptor outer segments where phototransduction occurs. Recombinant and native GCAP1 activate recombinant human retGC (outer segment-specific GC) and endogenous GC(s) in rod outer segment (ROS) membranes at low calcium. In addition, we isolate and clone a retinal homolog, termed GCAP2, that shows 50% identity with GCAP1. Like GCAP1, GCAP2 activates photoreceptor GC in a calcium-dependent manner. Both GCAP1 and GCAP2 presumably act on GCs by a similar mechanism; however, GCAP1 specifically localizes to photoreceptor outer segments, while in these experiments GCAP2 was isolated from extracts of retina but not ROS. These results demonstrate that GCAP1 is an activator of ROS GC, while the finding of a second activator, GCAP2, suggests that a similar mechanism of GC regulation may be present in outer segments, other subcellular compartments of the photoreceptor, or other cell types.

Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein

The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S‐modulin, the CAR (cancer‐associated retinopathy) protein and recoverin, the Ca2+‐dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.

Expression of GCAP 1 and GCAP2 in the retinal degeneration (rd) mutant chicken retina

We cloned the guanylate cyclase activating proteins, GCAP1 and GCAP2, from chicken retina and examined their expression in normal and predegenerate rd/rd chicken retina. Northern analyses show that the amounts of the single transcripts encoding GCAPI and GCAP2 are reduced to about 70% of normal levels in rdlrd retina. Western analyses reveal that GCAP2 levels appear normal in this retina, while GCAPI levels are reduced by more than 90%. The specific downregulation of GCAPI in rd/rd retina is consistent with a model for this disease in which activation of guanylate cyclase in the photoreceptors is abnormal, resulting in low levels of cGMP and an absence of phototransduction.

Expression of photoreceptor cyclic nucleotide-gated cation channel α subunit (CNGCα) in the liver and skeletal muscle

Glucagon and β‐adrenergic agents increase cAMP levels and stimulate Ca2+ influx in liver cells. There is no consensus as to the mechanism by which these hormones stimulate the influx of Ca2+. Using mouse retinal rod CNGCα cDNA probes, we cloned rat liver and skeletal muscle, and human hepatic CNGCα subunit sequences showing 97–100% identity with the human rod channel. In order to assess channel activity, the effect of cyclic nucleotides on free intracellular Ca2+ levels of isolated hepatocytes was measured. Dibutyryl‐cAMP was more effective in increasing free Ca2+ levels than dibutyryl‐cGMP. These data indicate that the CNGCα subunit is expressed in the both liver and skeletal muscle possibly mediating hormonal effects on ion fluxes.

Structure and Ca2+ regulation of frog photoreceptor guanylate cyclase, ROS-GC1.

Rod outer segment membrane guanylate cyclase (ROS-GC) is a critical component of the vertebrate phototransduction machinery. In response to photoillumination, it senses a decline in free Ca2+ levels from 500 to below 100 nM, becomes activated, and replenishes the depleted cyclic GMP pool to restore the dark state of the photoreceptor cell. It exists in two forms, ROS-GC1 and ROS-GC2. In outer segments, ROS-GCs sense fluctuations in Ca2+ via two Ca2+-binding proteins, which have been termed GCAP1 and GCAP2. In the present study we report on the cloning of two ROS-GCs from the frog retinal cDNA library. These cyclases are the structural and functional counterparts of the mammalian ROS-GC1 and ROS-GC2. There is, however, an important difference between the regulation of mammalian and frog ROS-GC1: In contrast to the mammalian, the frog form does not require the myristoylated form of GCAP1 for its Ca2+-dependent modulation. This feature is not dependent upon the ability of frog GCAP1 to bind Ca2+ because unmyristoylated GCAP1 mutants which do not bind Ca2+, activate frog ROS-GC1. The findings establish frog as a suitable phototransduction model and show a facet of frog ROS-GC signaling, which is not shared by the mammalian form.