Isolation and characterization of visual pigment kinase-related genes in carp retina: polyphyly in GRK1 subtypes, GRK1A and 1B.

Purpose: Visual pigment is phosphorylated and inactivated after light stimulus. The responsible enzyme is known as rhodopsin kinase or G-protein-coupled receptor kinase 1 (GRK1) in rods. We recently showed that the kinase in cones (GRK7) has much higher activity than GRK1 in rods in carp retina. During the course of these studies, we realized that there are several subtypes of GRK1 and GRK7. In the present study, therefore, to identify the GRK1 and GRK7 subtypes expressed in carp photoreceptors, we determined their nucleotide sequences together with their expression patterns in carp retina. We also analyzed their relationships to other GRK1s and GRK7s phylogenetically.

Methods: Oligonucleotides corresponding to the amino acid sequences conserved in GRK1 or GRK7 were synthesized to screen the GRK subtypes in a carp retinal cDNA library. The isolated partial cDNAs were used to determine the full length of GRK subtypes. Genomic Southern hybridization was performed to learn whether each of the isolated GRKs is encoded by a single gene or it is an allelic variation. Tissue localization of the isolated GRKs was examined with in situ hybridization.

Results: A novel subtype of GRK1, GRK1B, was found in addition to the conventional GRK1 (called GRK1A subtype in this study) in carp retina. The GRK1A subtype, more specifically the GRK1A-1 subtype, which is related to the mammalian-type GRK1, was expressed in rods, while the GRK1B subtype, related to chicken GRK1, was expressed in cones. Since GRK7-1 was also expressed in cones, carp cones express both GRK7-1 and GRK1B. There were two paralogous genes in all of the GRK1 and GRK7 subtypes in carp retina: GRK1A-1a and 1A-1b, GRK1Ba and 1Bb, and GRK7-1a and 7-1b. Each of these genes was suggested to be encoded by a single gene in the carp genome, and each pair was found to be expressed in the same type of photoreceptors.

Conclusions: Carp rods and cones express at least two kinds of visual pigment kinases. Phylogenetic analysis suggested that GRK7-1 together with GRK1A-1 and GRK1B appeared before divergence of vertebrates and that some of these genes were lost during evolution in a species-dependent manner. This evolutional process probably explains why the expression pattern of GRK1 and GRK7 is complex among vertebrate species.