Results Expression of FuU-Length and Truncated TGFc ~ Precursors in CHO Cells

A variety of growth factors including transforming growth factor-c~ (TGF-o0 are synthesized as transmembrane precursors. The short cytoplasmic domain of the transmembrane TGF-ot precursor lacks any apparent motif associated with signal transduction. However, the sequence conservation of this cytoplasmic domain and its abundance of cysteine residues, reminiscent of the cytoplasmic domains of CD4 and CD8, suggest a biological function. In this study, we showed that transmembrane TGF-c~ was rapidly internalized after interaction with a specific antibody and that this internalization was greatly decreased when the COOH-terminal 31 amino acids were removed. Chemical cross-linking experiments revealed two associated proteins of 86 and 106 kD which coimmunoprecipitated with the TGF-cx precursor. The association of p86 was dependent on the presence of the COOH-terminal cytoplasmic 31 amino acids of the TGF-ot precursor, whereas p106 still remained associated when this segment was deleted. In addition, p106 was tyrosine-phosphorylated and exposed on the cell surface. The protein complex associated with transmembrane TGF-ot displayed kinase activities towards tyrosine, serine, and threonine residues. These activities were not associated with transmembrane TGF-c~ when the COOH-terminal segment was truncated. The association of a protein kinase complex with transmembrane TGF-cx may provide the basic elements for a "reverse" mode of signaling through the cytoplasmic domain of this growth factor, which may lead to two-directional communication during ligandreceptor interaction. T GF-~x is synthesized as a transmembrane precursor (Derynck ot ai., 1984; Lee et al., 1985) and specific proteolytic cleavages in the extracellular domain produce soluble TGF-cx (Bringrnan et al., 1987; Gentry et al., 1987). However, the uncleaved transmembrane TGF-ot precursor often represents the predominant species in TGF-ot producing cells (Derynck, 1992). TGF-c~ is synthesized by many normal ceils and tissues, predominantly various epithelia including skin, brain (Coffey et al., 1987; Wilcox and Derynck, 1988a,b; Derynck, 1992), and tumor cells (for review see Derynck, 1992). TGF-a binds to the same receptor as EGF as a result of structural similarity (Massagut, 1983; Marquardt et al., 1984). Other members of the EGF/TGF-cx family also exist as cleavable transmembrane growth factors. Besides EGF and TGF-o~, this family includes amphiregulin (Shoyab et al., 1989; Plowman et al., 1990), HB-EGF (Higashiyama et al., 1991, 1992), betacellulin (Shing et al., 1993), and vaccinia virus growth factor Address all correspondence to Dr. R. Derynck, Departments of Growth and Development, Anatomy, University of California at San Francisco, San Francisco, CA 94143-0640. Dr. Steven A. Reeves's current address is Laboratory of Molecular Neuro-oncology, Massachusetts General Hospital, Charlestown, MA 02129. (Stroobant et al., 1985), all of which bind to the EGF/TGF-c~ receptor. In addition, the differentially spliced forms of neuregulins (Holmes et al., 1992; Wen et ai., 1992; Falls et al., 1993; Marchionni et al., 1993), lin 3 in Caenorhabditis elegans (Hill and Sternberg, 1992) and spitz (Rutledge et al., 1992), and gurken (Neumann-Silberberg and Schiipbach, 1993) gene products in Drosophila also belong to this family. The cytoplasmic domain of the TGF-~x precursor is only 39 amino acids long, contains 7 cysteines, and is highly conserved among species (Derynck et al., 1984; Lee et al., 1985; Zurfluh et al., 1990; Chiang et al., 1991), thus suggesting a defined biological function. However, examination of this sequence does not reveal any known functional motifs. Recently, it has been demonstrated that the COOH-terminal valines in the TGF-, precursor are required for efficient cleavage of the ectodomain of transmembrane TGF-o~ and subsequent release of soluble TGF-c~ (Bosenberg et ai., 1992). Since the short cytoplasmic domain of TGF-tx is unlikely to have enzymatic activity, its potential function may be mediated through proteins that interact with the cytoplasmic domain which may associate noncovalently through the cysteine-rich regions. This is modeled after the T cell membrane glycoproteins CIM and CD8, which have short cytoplasmic domains that interact with the associated lck kinase © The Rockefeller University Press, 0021-9525194105/903/14 $2.00 The Journal of Cell Biology, Volume 125, Number 4, May 1994 903-916 903 on F ebuary 1, 2013 jcb.rress. org D ow nladed fom Published May 15, 1994