Regulation of rat AMP deaminase 3 (isoform C) by development and skeletal muscle fibre type

Abstract

AMP deaminase (AMPD) is characterized by a multigene family in rodents and man. Highly conserved rat and human AMPD1 and AMPD2 genes produce protein products that exhibit cross-species immunoreactivities (AMPD1, rat isoform A and human isoform M; AMPD2, rat isoform B and human isoform L). A third gene, AMPD3, has been described in humans, but antisera raised against its purified protein product (isoform E) reportedly does not cross-react with a third activity purified from rat tissues (isoform C). This study was designed to address this latter issue by cloning, sequencing and expressing rat AMPD3 cDNA species. Similarly to the human AMPD3 gene, the rat AMPD3 gene produces multiple transcripts that differ at or near their 5′ ends. The boundary at which these alternative sequences diverge is precisely conserved in both species. Across the region that is common to all rat and human AMPD3 cDNA species, nucleotide and predicted amino acid sequences are 89% and 93% identical respectively, although the rat open reading frame is lacking two separate in-frame codons in the 5′ end. Extreme 5′ regions between the two species are entirely divergent, and one alternative rat sequence is predicted to confer at least 36 additional N-terminal residues to its encoded AMPD3 polypeptide. A comparison of 3′ untranslated regions indicates that the rat sequence is 250 bp longer and contains multiple consensus polyadenylation signals. Examination of relative rat AMPD3 gene expression shows (1) variable patterns of alternative mRNA abundance across adult tissues, (2) developmental regulation in skeletal muscle and liver, and (3) greater mRNA abundance in adult red (soleus) than in mixed (plantaris) and white (outer gastrocnemius) skeletal muscle. Finally, baculoviral expression of rat and human AMPD3 proteins produces enzymes that are chromatographically and kinetically similar. Moreover, both recombinant activities immunoreact with anti-C and anti-E serum. These combined results demonstrate that rat isoform C and human isoform E are homologous cross-species AMPD3 proteins.