Cloning and tissue expression of eleven troponin-C isoforms in the American lobster, Homarus americanus.

Troponin-C is the Ca(2+)-binding subunit of the troponin regulatory complex in striated muscles. As TnC isoforms can influence the Ca(2+)-activation properties of fiber phenotypes, the diversity and tissue distribution of TnC cDNAs were assessed in the American lobster, Homarus americanus. We cloned ten full-length cDNAs and one partial cDNA coding for distinct TnC isoforms. Five were sequenced from expressed sequence tag clones and were designated Ha-TnC(2b)(') (2094 nt, 141 aa and 15.9 kDa), -C(4)(') (1667 nt, 155 aa and 17.3 kDa), -C(5) (2884 nt, 149 aa and 17.3 kDa), -C(6) (2439 nt, 155 nt and 17.4 kDa) and-C(6x) (2171 nt, 154 aa and 16.9 kDa). The remainder were cloned using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends: five full-length cDNAs, designated Ha-TnC(1) (814 nt, 150 aa and 17.1 kDa), -C(2a) (639 nt, 152 aa and 17.2 kDa), -C(2b)('') (2136 nt, 155 aa and 17.5 kDa), -C(3) (1046 nt, 150 aa and 16.9 kDa), -C(4)('') (842 nt, 108 aa and 12.1 kDa) and one partial (3') cDNA, designated Ha-TnC(4)(''') (563 nt and 57 aa). Ha-TnC(1), -C(2a), and-C(2b)(') corresponded to lobster TnC sequences in the GenBank protein database (Ha-TnC(1), -C(2a), and-C(2b)). Alternative splicing appeared responsible for TnC(2b)(') and-C(2b)(''); TnC(4)('), -C(4)('') and-C(4)('''); and TnC(6) and-C(6x). The deduced amino acid sequences differed primarily in the terminal regions and EF-hands I and III. Ha-TnC(6x) had a highly divergent 76 aa proline-rich N-terminal sequence. Tissue expression of the Ha-TnC isoforms was analyzed qualitatively by endpoint PCR. Ha-TnC(1), -C(2a), -C(2b)('), -C(2b)('') and-C(3) were expressed primarily in skeletal muscles; Ha-TnC(5) was expressed in heart; and Ha-TnC(4) and-C(6) variants were expressed in muscles and other tissues. The number and diversity of TnC sequences suggest the potential for varying the Ca(2+)-activated properties of the troponin-tropomyosin regulatory complex through differential expression of TnC isoforms.