Genomic and cDNA clones encoding portions of a putative catfish parathyroid hormone (PTH) 2 receptor (PTH2R) led to the isolation of a cDNA encoding a fulllength zebrafish PTH2R (zPTH2R). The zPTH2R shared 63 and 60% amino acid sequence identity with human and rat PTH2Rs, respectively, 47-52% identity with mammalian and frog PTH/PTHrP receptors (PTH1R), and less than 37% with other members of this family of G protein-coupled receptors. COS-7 cells expressing zPTH2R (43) . FuguPTHrP also failed to activate the human PTH2R but had similar efficiency and efficacy as hPTH and hPTHrP when tested with cells expressing the human PTH1R. Agonist-dependent activation of zPTH2R was less efficient than that of zPTH2R(43), and both receptor variants showed no cAMP accumulation when stimulated with either secretin, growth hormone-releasing hormone, or calcitonin. The zPTH2R thus has ligand specificity similar to that of the human homolog, which raises the possibility that a PTH-like molecule exists in zebrafish, species which lack parathyroid glands.
Sequence analysis of H chain cDNA derived from the spleen of an individual catfish has shown that somatic mutation occurs within both the VH- and JH-encoded regions. Somatic mutation preferentially targets G and C nucleotides with approximately balanced frequencies, resulting in the predominant accumulation of G-to-A and C-to-T substitutions that parallel the activation-induced cytidine deaminase nucleotide exchanges known in mammals. The overall mutation rate of A nucleotides is not significantly different from that expected by sequence-insensitive mutations, and a significant bias exists against mutations occurring in T. Targeting of mutations is dependent upon the sequence of neighboring nucleotides, allowing statistically significant hotspot motifs to be identified. Dinucleotide, trinucleotide, and RGYW analyses showed that mutational targets in catfish are restricted when compared with the spectrum of targets known in mammals. The preferential targets for G and C mutation are the central GC positions in both AGCT and AGCA. The WA motif, recognized as a mammalian hotspot for A mutations, was not a significant target for catfish mutations. The only significant target for A mutations was the terminal position in AGCA. Lastly, comparisons of mutations located in framework region and CDR codons coupled with multinomial distribution studies found no substantial evidence in either independent or clonally related VDJ rearrangements to indicate that somatic mutation coevolved with mechanisms that select B cells based upon nonsynonymous mutations within CDR-encoded regions. These results suggest that the principal role of somatic mutation early in phylogeny was to diversify the repertoire by targeting hotspot motifs preferentially located within CDR-encoded regions.
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