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Dimerization of Kit-ligand and efficient cell-surface presentation requires a conserved Ser-Gly-Gly-Tyr motif in its transmembrane domain

Frédérique Paulhe^*,1, Monique Wehrle-Haller^*,1, Marie-Claude Jacquier^*, Beat A. Imhof, Séverine Tabone-Eglinger^*, and Bernhard Wehrle-Haller^*,2

^* Department of Cellular Physiology and Metabolism and

Department of Pathology and Immunology, Centre Médical Universitaire, University of Geneva, Geneva, Switzerland; and

Cytokine and Cancer Group, INSERM U590, Centre Léon Bérard, University Claude Bernard, Lyon, France

² Correspondence: Department of Cellular Physiology, Centre Médical Universitaire, 1.Rue Michel-Servet, 1211 Geneva 4, Switzerland. E-mail: bernhard.wehrle-haller{at}unige.ch

Kit-ligand (Kitl), also known as stem cell factor, is a membrane-anchored, noncovalently bound dimer signaling via the c-kit receptor tyrosine kinase, required for migration, survival, and proliferation of hematopoietic stem and germ cells, melanocytes, and mastocytes. Despite its fundamental role in morphogenesis and stem cell biology, the mechanisms that regulate Kitl dimerization are not well understood. By employing cell-permeable cross-linker and quantitative bimolecular fluorescence complementation of wild-type and truncated forms of Kitl, we determined that Kitl dimerization is initiated in the endoplasmic reticulum and mediated to similar levels by the transmembrane and the extracellular growth factor domain. Further biochemical and mutational analysis revealed a conserved Ser-Gly-Gly-Tyr-containing motif that is required for transmembrane domain dimerization and efficient cell-surface expression of Kitl. A novel intracellular capture assay with the Kitl transmembrane domain as bait revealed specific interactions with Kitl, but not with unrelated transmembrane proteins. During evolution, the transmembrane dimerization motif appeared in Kitl at the transition from teleosts to tetrapods, which correlates with the emergence of Kitl as a supporter of stem cell populations. Thus, transmembrane-mediated association of membrane-anchored growth factors consists of a novel mechanism to improve paracrine signaling and morphogenesis.—Paulhe, F., Wehrle-Haller, M., Jacquier, M.-C., Imhof, B. A., Tabone-Eglinger, S., Wehrle-Haller, B. Dimerization of Kit-ligand and efficient cell-surface presentation requires a conserved Ser-Gly-Gly-Tyr motif in its transmembrane domain.

Key Words: bimolecular fluorescence complementation • growth factors • intracellular transport • c-Kit • evolution • Flt3-L