Molecular cloning and nucleotide sequence of a cDNA encoding recoverin from human retina

In the process of photoreceptor signal transduction, light initiates an enzymatic cascade that leads to hydrolysis of cyclic GMP (cGMP) and closure of cGMP-gated sodium-calcium channels resulting in photoreceptor hyperpolarization. Recoverin is a calcium-binding protein that is thought to reverse the effects of light on cGMP levels by activating guanylate cyclase. Guanylate cyclase produces cGMP to overcome the cGMP-hydrolysing effect of phosphodiesterase, and reopens the sodium-calcium channels in photoreceptor outer segments. We have cloned and sequenced a cDNA encoding recoverin in human retina. The human nucleotide sequence is 88% identical to the bovine sequence, and contains a 600-base pair (bp) open reading frame encoding 200 amino acids. In situ hybridization of cultured Y79 human retinoblastoma cells with a radioactive recoverin cDNA probe showed intense, specific labeling of the cytoplasm, indicating the presence of mRNA encoding recoverin. Direct sequencing of a Y79 retinoblastoma cDNA polymerase chain reaction (PCR) product confirmed the presence of recoverin in this human cell line.