Insulin-like peptide 6: characterization of secretory status and posttranslational modifications.
Insulin-like peptide 6 (Insl6) is a member of the insulin/relaxin superfamily with unknown biological function(s). In the current report, we establish that meiotic and postmeiotic germ cells of the testis are the principal sites of expression of Insl6. Analysis of stably or transiently transfected cells revealed that Insl6 is a secreted protein localized to the endoplasmic reticulum and Golgi. Secretion could be detected in both CHO and GC2 germ cells and was sensitive to brefeldin A treatment. In cell lysates, the predominant Insl6 band was approximately 28 kDa in size. In contrast, the predominant Insl6 species in the supernatant was 8 kDa in size, suggesting posttranslational processing of the precursor protein. Ectopically expressed Insl6 is processed and secreted in furin-deficient LoVo cells and in CHO cells treated with a furin inhibitor, although the size profile of the secreted protein is altered suggesting that Insl6 is a substrate for furin action. Furthermore, mutation of a putative furin cleavage site in the Insl6 peptide resulted in aberrant processing of the Insl6 peptide. Additional investigations of the structure of Insl6 protein provided evidence for posttranslational modifications of Insl6, including the presence of disulfide bonds, glycosylation, and ubiquitination. On the basis of the demonstrated secretory status of Insl6, we speculate that the physical proximity of the germ cell to the Sertoli cell renders the Sertoli cell a likely candidate for Insl6 action.
Lu
Chunxia
C
Department of Pediatrics, University of Michigan, 1205 Medical Professional Building Box 0718, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718, USA.
Walker
William H
WH
Sun
Jinhong
J
Weisz
Ora A
OA
Gibbs
Robert B
RB
Witchel
Selma F
SF
Sperling
Mark A
MA
Menon
Ram K
RK
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry; D005260:Female; D045683:Furin / Q000502:physiology; D018507:Gene Expression Regulation, Developmental / Q000528:radiation effects; D005854:Germ Cells / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D008969:Molecular Sequence Data; D011247:Pregnancy; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley; D013737:Testis / Q000196:embryology / Q000378:metabolism / Q000528:radiation effects; D014018:Tissue Distribution; D025801:Ubiquitin / Q000378:metabolism
D004220:Disulfides; C512664:Insl6 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D025801:Ubiquitin; D045683:Furin