The proto‐oncogene c‐kit encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand and is allelic with the murine white‐spotting locus (W). Mutations at the W locus affect various aspects of hematopoiesis, the proliferation and migration of primordial germ cells and melanoblasts during development. The original W mutation and W37 are severe lethal mutations when homozygous. In the heterozygous state the W mutation has a weak phenotype while W37 has dominant characteristics. Wv and W41 are weak W mutations with dominant characteristics. We have characterized the molecular basis of these four W mutations and determined their effects on mast cell differentiation by using a fibroblast/mast cell co‐culture assay. We show that W37, Wv and W41 are the result of missense mutations in the kinase domain of the c‐kit coding sequence (W37 E‐‐‐‐K at position 582; Wv T‐‐‐‐M position 660 and W41 V‐‐‐‐M position 831), which affect the c‐kit associated tyrosine kinase to varying degrees. The c‐kit protein products in homozygous mutant mast cells are expressed normally, although the 160 kd cell membrane form of the c‐kitW37 protein displays accelerated turnover characteristics. The W mutation is the result of a 78 amino acid deletion which includes the transmembrane domain of the c‐kit protein. A 125 kd c‐kit protein was detected in homozygous W/W mast cells which lacks kinase activity and is not expressed on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

The pleiotropic effects of the Kit receptor system are mediated by Kit‐Ligand (KL) induced receptor autophosphorylation and its association with and activation of distinct second messengers, including phosphatidylinositol 3′‐kinase (PI3‐kinase), p21ras and mitogen‐activated protein kinase (MAPK). To define the role of PI3‐kinase, p21ras and MAPK in Kit‐mediated cell proliferation, survival and adhesion in bone marrow‐derived mast cells (BMMC), mutant Kit receptors were expressed in Wsh/Wsh BMMC lacking endogenous c‐kit expression. The introduction of both murine Kit(S) and KitL (isoform containing a four amino acid insert) into Wsh/Wsh BMMC restored KL‐induced proliferation, survival and adhesion to fibronectin, as well as activation of PI3‐kinase, p21ras and MAPK, and induced expression of c‐fos, junB, c‐myc and c‐myb mRNA. Substitution of tyrosine 719 in the kinase insert with phenylalanine (Y719F) abolished PI3‐kinase activation, diminished c‐fos and junB induction, and impaired KL‐induced adhesion of BMMC to fibronectin. In addition, the Y719F mutation had partial effects on p21ras activation, cell proliferation and survival, while MAP kinase activation was not affected. On the other hand, Y821F substitution impaired proliferation and survival without affecting PI3‐kinase, p21ras and MAPK activation, and induction of c‐myc, c‐myb, c‐fos and c‐jun mRNA, while KL‐induced cell adhesion to fibronectin remained intact. In agreement with a role for PI3‐kinase in Kit‐mediated cell adhesion, wortmannin blocked Kit‐mediated cell adhesion at concentrations known to specifically inhibit PI3‐kinase. We conclude, that association of Kit with p85PI3‐K, and thus with PI3‐kinase activity, is necessary for a full mitogenic as well as adhesive response in mast cells. In contrast, tyrosine 821 is essential for Kit‐mediated mitogenesis and survival, but not cell adhesion.

The murine white spotting locus (W) is allelic with the proto-oncogene c-kit, which encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand. Mutations at the W locus affect various aspects of hematopoiesis and the proliferation and migration of primordial germ cells and melanoblasts during development to varying degrees of severity. The W42 mutation has a particularly severe effect in both the homozygous and the heterozygous states. The molecular basis of the W42 mutation was determined. The c-kit protein products in homozygous mutant mast cells were expressed normally but displayed a defective tyrosine kinase activity in vitro. Nucleotide sequence analysis of mutant complementary DNAs revealed a missense mutation that replaces aspartic acid with asparagine at position 790 in the c-kit protein product. Aspartic acid-790 is a conserved residue in all protein kinases. These results provide an explanation for the dominant nature of the W42 mutation and provide insight into the mechanism of c-kit-mediated signal transduction.

The extracellular region of the human interleukin-10 (hIL-10) receptor was expressed using a myeloma cell line and was purified to homogeneity by ligand-affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis indicated that the soluble receptor is glycosylated and has an apparent molecular mass of 35,000-45,000. Under native conditions, soluble hIL-10 receptor was determined by gel filtration to be a monomeric protein. Soluble hIL-10 receptor was able to inhibit the binding of 125I-hIL-10 to the full-length receptor and was able to antagonize the effect of human IL-10 in cell proliferation and cytokine synthesis inhibition. The apparent dissociation constant (Kd) of soluble hIL-10 receptor was determined to be 563 +/- 59 pM, approximately 2- to 10-fold higher than that found on intact cells (Tan, J. C., Indelicato, S. R., Narula, S. K., Zavodny, P. J., and Chou, C.-C. (1993) J. Biol. Chem. 268, 21053-21059; Liu, Y., Wei, S. H.-Y., Ho, A. S.-Y., de Waal Malefyt, R., and Moore, K. W. (1994) J. Immunol. 152, 1821-1829). When hIL-10 binds soluble hIL-10 receptor in solution, a single complex was detected by gel filtration, and the complex was found to consist of two hIL-10 dimers and four soluble receptor monomers, suggesting that hIL-10 may induce a novel mode of oligomerization of the receptor upon binding.

Human natural killer (NK) cells are large granular lymphocytes that constitutively express functional forms of the interleukin-2 receptor (IL-2R) and lyse tumor and virally infected cells without prior sensitization. NK cells with high density expression of CD56 (CD56bright) express the high affinity IL-2R and proliferate in response to low (picomolar) concentrations of IL-2. CD56dim NK cells express the intermediate affinity IL-2R and demonstrate enhanced cytotoxic activity without proliferation in response to high (nanomolar) concentrations of IL-2. In the present study, we characterized IL-10R expression on human NK cells and the functional consequences of IL-10 binding directly to highly purified subsets of CD56bright and CD56dim NK cells. Binding studies using 125I-IL-10 indicated that resting human NK cells constitutively express the IL-10 receptor protein at a surface density of approximately 90 receptor sites per cell, with a kd of approximately 1 nmol/L. Alone, IL-10 did not induce proliferation of CD56bright or CD56dim NK cell subsets. However, at low concentrations (0.5 to 5 ng/mL), IL-10 significantly augmented IL-2-induced proliferation of the CD56bright NK cell subset mediated via the high-affinity IL-2R. In the absence of IL-2, IL-10 was able to induce significant NK cytotoxic activity against NK-resistant tumor cell targets in both subsets of NK cells in a dose-dependent fashion. Furthermore, the combination of IL-10 and IL-2 had an additive effect on NK cytotoxic activity, whereas that of IL-10 and IL-12 did not. Production of interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor by IL-2-activated NK cells was also significantly enhanced by IL-10. Neither resting nor activated human NK cells appear to produce human IL-10 protein. In summary, NK cells constitutively express the IL-10R protein in low density, and the functional consequences of IL-10 binding directly to human NK cell subsets appear to be stimulatory and dose-dependent. In contrast to its direct effects on human T cells and monocytes/macrophages, IL-10 potentiates cytokine production by human NK cells.