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Receptor dimerization is ubiquitous to the action of all receptor tyrosine kinases, and in the case of dimeric ligands, such as the stem cell factor (SCF), it was attributed to ligand bivalency. However, by using a dimerization-inhibitory monoclonal antibody to the SCF receptor, we confined a putative dimerization site to the nonstandard fourth immunoglobulin-like domain of the receptor. Deletion of this domain not only abolished ligand-induced dimerization and completely inhibited signal transduction, but also provided insights into the mechanism of the coupling of ligand binding to dimer formation. These results identify an intrinsic receptor dimerization site and suggest that similar sites may exist in other receptors.

Mast cell growth factor (MGF) (also called stem cell factor) synergizes with several lymphokines, including interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), to promote proliferation and differentiation of certain hemopoietic progenitor cells. Although similar patterns of tyrosine-phosphorylated proteins characterize cells stimulated by MGF, IL-3, and GM-CSF, only the MGF receptor is a tyrosine kinase, and the heterodimeric receptors for IL-3 and GM-CSF share a common beta subunit that is devoid of enzymatic activity. Here we show that signaling pathways utilized by all three cytokines include the cytoplasmic tyrosine kinase JAK2. Analysis of several factor-dependent myeloid cell lines indicated that JAK2 is physically associated with the common beta subunit and with MGF receptor (c-Kit) even prior to ligand binding. However, each of the ligands induced elevated tyrosine phosphorylation of JAK2 and a consequent increase in its catalytic activity. These results demonstrate for the first time the convergence within the same myeloid cells of signaling pathways originating in two distinct lymphokine receptors and a tyrosine kinase receptor on activation of a cytoplasmic tyrosine kinase.

The mast cell growth factor (MGF) affects migration, proliferation and differentiation of erythroid and myeloid progenitor cells by binding to a transmembrane receptor tyrosine kinase encoded by the c-Kit proto-oncogene. By using MGF-dependent human myeloid cell lines (M-07e and TF-1), here we show that a Kit-related 100 kDa protein is associated with the cell but it undergoes release into the medium upon treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C. Immunological analysis with a series of antibodies to Kit indicated that the released protein (p100Kit) contains the whole glycosylated extracellular portion of the transmembrane Kit protein (p145Kit). The secreted protein retained the ability to specifically bind MGF. Moreover, p100Kit was able to block the mitogenic effect of MGF on cultured M-07e cells, suggesting that the soluble protein may function as a physiological antagonist of MGF.

Despite extensive research on the molecular mechanisms of signal transduction by growth factors and their oncogenic receptor tyrosine kinases, the physiological relevance of these pathways, especially in mammals, remains largely unknown. A unique exception is the Steel factor (SLF) and its c-kit-encoded receptor, because many natural germ line mutations of both the ligand and the receptor exist in mice. The protooncogene c-kit encodes a cell surface receptor that belongs to the immunoglobulin gene family and carries an intrinsic tyrosine kinase activity in its cytoplasmic portion. The precursor of the Kit ligand, SLF, is also a transmembrane protein that exists as a soluble factor as well as a cell surface protein. The interaction of Kit with SLF leads to receptor dimerization, kinase activation, and tyrosine phosphorylation of cytoplasmic proteins that contain Src homology 2 motifs. Various mutations in Kit and SLF result in a defective signaling pathway and underly the complex phenotypes of W and Sl mice, respectively. The early development of at least four cell lineages is affected. These are erythrocytes, melanocytes, germ cells, and mast cells. Correlation between the behavior of these lineages and specific mutations uncovered interesting physiological aspects of the mechanism of signal transduction by a polypeptide growth factor. These include the different degrees of severity of affected lineages, indications for distinct functions during early embryonic development and at late phases, the significance of synergy between a growth factor and lymphokines, the interaction between mutant and wild-type proteins in heterozygous animals, and the possibility that a surface-anchored ligand may act differently than a soluble factor. Predictably, the lessons learned with Kit and Sl mice will be widely relevant to other pairs of ligands and receptors that control the function of different cell lineages and physiological processes.

Binding of the Steel factor (SLF) to the product of the c-kit proto-oncogene stimulates the receptor's intrinsic tyrosine kinase that phosphorylates a set of cytoplasmic signaling molecules. Germ-line mutations in the genes that encode the receptor or the ligand result in remarkably similar phenotypes that affect melanogenesis, erythropoiesis and gametogenesis in mice. We concentrated on the initial events of the signal transduction pathway that underlies these processes. The extracellular portion of Kit is comprised of five immunoglobulin-(Ig)-like domains. Ligand binding to this domain induces rapid and extensive dimerization of the receptor molecules in a mechanism that involves monovalent binding of the dimeric ligand, followed by an increase in receptors' affinity and gradual stabilization of the dimers. It thus appears that Kit has at least two functions: ligand binding and ligand-induced receptor dimerization, in addition to the kinase activity. Both functions are independent of the transmembrane and cytoplasmic domains, as a recombinant soluble ectodomain retained high affinity to SLF and ligand-dependent dimerization. In order to correlate these functions with specific structures, we employed ligand-competitive monoclonal antibodies, soluble deletion mutants of the ectodomain and chimeric human-mouse Kit proteins. These approaches indicated that the N-terminal three Ig-like domains constitute the binding site, whose core is the second domain. Further experiments suggested that a putative dimerization site is distinct from the binding cleft and may be located on the fourth Ig-like domain.