Abstract
The ADAM (A Disintegrin And Metalloprotease) family of cell-surface proteins may have an important role in cellular interactions and in modulating cellular responses. In this report we describe a novel, secreted form of human ADAM 12 (meltrin alpha), designated ADAM 12-S (S for short), and a larger, membrane-bound form designated ADAM 12-L (L for long form). These two forms arise by alternative splicing of a single gene located on chromosome 10q26. Northern blotting demonstrated that mRNAs of both forms are abundant in human term placenta and are also present in some tumor cell lines. The ADAM 12-L transcript can also be detected in normal human adult skeletal, cardiac, and smooth muscle. Human A204 embryonal rhabdomyosarcoma cells that do not differentiate into muscle cells and do not express any form of ADAM 12 were stably transfected with an ADAM 12-S minigene encoding the disintegrin domain, the cysteine-rich domain, and the unique 34 amino acid carboxyl terminus. Nude mouse tumors derived from these transfected cells contained ectopic muscle cells of apparent mouse origin as shown by species-specific markers. These results may have potential applications in the development of muscle-directed gene and cell therapies.
Highlights
ADAMs1 are a recently discovered family of membrane-anchored cell-surface proteins
Full-length snake venom metalloproteases (SVMPs) are processed to generate a metalloprotease, which is able to degrade proteins of the basement membrane such as type IV collagen and laminin [5], and a disintegrin domain, which can inhibit the function of platelets by interacting with platelet integrin GPIIb-IIIa [8]
Evidence for a role in muscle cell fusion was provided by studies showing that transfection of mouse C2 cells with a minigene of adam 12 lacking the pro- and metalloprotease domains accelerated cell fusion, whereas antisense constructs blocked myoblast fusion
Summary
Isolation and Sequencing of Human ADAM 12 cDNA Clones—A positive prey clone (S1) was isolated from a human yeast two-hybrid placental cDNA library (CLONTECH catalog number HL4025AH) us-. A DNA fragment coding for the disintegrin domain, cysteine-rich domain, and the unique carboxyl terminus of ADAM 12-S was prepared by PCR amplification using the ADAM 12-S cDNA plasmid as a template and the following primers: 5Ј-dCCAAAGCTTGAAGTCAGGGAGTCTTTC and 5Ј-dCCATCTAGATCAGATGAGTGTCAGTGA. The 987-bp PCR product contained nt 1560 –2528 of ADAM 12-S, with HindIII and XbaI cloning sites This fragment was inserted at the HindIII/XbaI sites of pSecTagB, yielding plasmid p1095, consisting of an ADAM 12 minigene driven by a cytomegalovirus promoter, fused to an Ig -chain leader sequence to allow secretion of the protein. Cells were transfected with an expression plasmid for a human ADAM 12-S minigene (p1095) or the expression vector with no cDNA insert (pSecTagB). A204 cells were stably transfected with an expression plasmid for a human ADAM 12-S minigene (p1095) or the expression vector with no cDNA insert (pSecTagB). RT-PCR was applied to examine for the presence of myf-5 transcript in cultured cells and nude mouse tumors using a Stratagene kit and primers specific for mouse myf-5 (5Ј-dCTCTCCCGATGATCACTCCT and 5ЈD-CCTGTAATGGATTCCAAGCTG), derived from GenBank number X56182 [35]
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