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 of five highly-related genes (GH-N, GH-V, PL-L, PL-A, PL-B). This evolutionarily young gene cluster codes for an array of mRNAs and proteins, such as the 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-Delta4, 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 testis. To elucidate the role of hPL-A2 in male reproduction and pregnancy, testicular PL-A2 cDNA was cloned in a complementary overlapping 2-way RT-PCR approach 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 RNA, but is a stable mRNA isoform generated by placental/testis-specific splicing 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 determined by hPL-specific monoclonal antibodies. In contrast, the C-terminus shares a hydrophobic region presumably responsible for membrane insertion. By the use of confocal microscopy recombinant hPL-A2 was localized in the cell membrane. Thus, hPL-A2 might exert its function by modulating GH/PL actions or act as an independent growth-regulatory molecule itself and its functions in male reproduction and embryonic development remain to be investigated.
Untergasser
G
G
Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020, Innsbruck, Austria.
Hermann
M
M
Rumpold
H
H
Pfister
G
G
Berger
P
P
D000368:Aged; D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000378:metabolism; D001483:Base Sequence; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D005260:Female; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007438:Introns; D008297:Male; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D010920:Placenta / Q000378:metabolism; D010928:Placental Lactogen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013737:Testis / Q000378:metabolism*; D013997:Time Factors
D000911:Antibodies, Monoclonal; D012333:RNA, Messenger; D011994:Recombinant Proteins; C415667:placental lactogen A-2; D019382:Human Growth Hormone; D010928:Placental Lactogen