An unusual member of the human growth hormone/placental lactogen (GH/PL) family, the testicular alternative splicing variant hPL-A2: recombinant expression revealed a membrane-associated growth factor molecule

The human growth hormone/placental lactogen (GH/PL) gene cluster consists o f five highly-related genes (GH-N, GH-V, PL-L, PL-A, PL-B). This evolutiona rily young gene cluster codes for an array of mRNAs and proteins, such as t he major 22 k forms (hGH-N/V, identical PL-A and B), 20 k and 17.5 k hGH-N and the recently described 25 k hGH-Delta 4, a presumably chimeric molecule . In addition, two longer alternatively spliced, (intron D retaining) mRNAs isoforms, termed PL-A2 and GH-V2, have been described in placenta and test is. To elucidate the role of hPL-A2 in male reproduction and pregnancy, tes ticular PL-A2 cDNA was cloned in a complementary overlapping 2-way RT-PCR a pproach to analyze translation, localization and structure/function of this unusual member of the GH/PL growth factor family. Analysis of insect mRNA revealed that intron D-retaining PL-A2 cDNA was expressed without splicing in the baculovirus expression system. Thus, PL-A2 mRNA does not represent a nuclear intermediate splicing product simply co-isolated with the mature R NA, but is a stable mRNA isoform generated by placental/testis-specific spl icing factors. Recombinant protein was present in whole cell extracts, and no secreted protein was detected in the supernatant. Immunologically, the N -terminus of the 230 amino acid protein is similar to 22 k hPL-A/B, as dete rmined by hPL-specific monoclonal antibodies. In contrast, the C-terminus s hares a hydrophobic region presumably responsible for membrane insertion. B y the use of confocal microscopy recombinant hPL-A2 was localized in the ce ll membrane. Thus, hPL-A2 might exert its function by modulating GH/PL acti ons or act as an independent growth-regulatory molecule itself and its func tions in male reproduction and embryonic development remain to be investiga ted. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.