Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic modulation of vascular smooth muscle cells.

The bio-effects of cellular repressor of E1A-stimulated genes (CREG) have been proposed to depend on its N-glycosylation and binding to mannose-6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R). The present study aimed to investigate the detailed mode and specific sites for their binding and the functional relevance of this binding in the phenotypic modulation of vascular smooth muscle cells (SMCs). Wild-type and glycosylation mutant human CREG (wtCREG and mCREG) proteins were expressed and isolated from HEK293 cells. CREG knocked-down SMCs were used to evaluate their biological activity. Both wtCREG and mCREG arrest cell cycle progression of CREG knocked-down SMCs when added to the culture medium. In vitro binding assay revealed that CREG bound to M6P/IGF2R extracellular domains 7-10 and 11-13 in a glycosylation-dependent and -independent manner, respectively. Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody suggest that the binding to domains 11-13, as well as to 7-10, is adequate for CREG to modulate SMC proliferation. These data suggest that soluble CREG protein can exert its biological function via glycosylation-independent binding to the extracellular domains 11-13 of cell surface M6P/IGF2R, and thereby provide novel insights into CREG modulation of SMC phenotypic switching from contractile to proliferative.