The secreted form of a melanocyte membrane-bound glycoprotein (Pmel17/gp100) is released by ectodomain shedding.
Ectodomain shedding is a proteolytic mechanism by which a transmembrane protein is converted into a secreted form. Pmel17/gp100 is a melanocyte-specific membrane-bound glycoprotein that has amyloid characteristics and forms fibrillar structures in melanosomes after a complex sequence of post-translational processing and trafficking events, including cleavage by a furin-like proprotein convertase (PC). A secreted form of Pmel17 (termed sPmel17) was also thought to be released due to cleavage by a PC. We used multidisciplinary approaches to demonstrate that sPmel17 is released by ectodomain shedding at the juxtamembrane and/or intramembrane motif and to show that this is independent of cleavage by a PC. We further show that sPmel17 consists of 2 fragments linked by disulfide bonds and that the shedding is inhibited at low temperature but not by metalloproteinase inhibitors. Moreover, treatment with a phorbol ester or a calmodulin inhibitor induces Pmel17 shedding. We also refine the reactivity of HMB50 and NKI/beteb, 2 monoclonal antibodies commonly used as melanoma-specific markers. The fact that those antibodies require physically separated domains of Pmel17 sheds interesting light on its 3-dimensional conformation. We conclude that sPmel17 is released by regulated proteolytic ectodomain shedding.-Hoashi, T., Tamaki, K., Hearing, V. J. The secreted form of a melanocyte membrane-bound glycoprotein (Pmel17/gp100) is released by ectodomain shedding.
Hoashi
Toshihiko
T
V.J.H., National Institutes of Health, Laboratory of Cell Biology, Bldg. 37, Rm. 2132, Bethesda, MD 20892, USA. thoashi-tky@umin.ac.jp
Tamaki
Kunihiko
K
Hearing
Vincent J
VJ
D000256:Adenoviridae; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002460:Cell Line; D045744:Cell Line, Tumor; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010703:Phorbol Esters / Q000494:pharmacology; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D011109:Polymethacrylic Acids / Q000494:pharmacology; D043484:Proprotein Convertases / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011817:Rabbits; D019810:Sodium Azide / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D058951:gp100 Melanoma Antigen
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D008562:Membrane Glycoproteins; C548637:PMEL protein, human; D010703:Phorbol Esters; D011109:Polymethacrylic Acids; D013449:Sulfonamides; D058951:gp100 Melanoma Antigen; C500651:poly(MPC-co-MA); D010767:Phosphorylcholine; C017967:W 7; D019810:Sodium Azide; D043484:Proprotein Convertases