Sensory and motor neuron-derived factor is a transmembrane heregulin that is expressed on the plasma membrane with the active domain exposed to the extracellular environment

Abstract

The β-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the β-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other β-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl- terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor.