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 proliferation of vascular smooth muscle cells

Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs 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 indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.

POST1/C12ORF49 regulates the SREBP pathway by promoting site-1 protease maturation

Sterol-regulatory element binding proteins(SREBPs)are the key transcriptional regulators of lipid metabolism.The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi,where it is sequentially cleaved by site-1 protease(S1P)and site-2 protease and releases a nuclear form to modulate gene expression.To search for new genes regulating cholesterol metabolism,we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease(POSH),encoded by C120RF49,is critically involved in the SREBP signaling.Ablation of POSH decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes.POSH binds S1P,which is synthesized as an inactive protease(form A)and becomes fully mature via a two-step autocatalytic process involving forms B/B and C/C.POSH promotes the generation of the functional S1P-C/C from S1P-B/B(canonical cleavage)and,notably,from S1P-A directly(non-canonical cleavage)as well.This POSH-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6,CREB3 family members and the a/p-subunit precursor of N-acetylglucosamine-1-phospho-transferase.Together,we demonstrate that POSH is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis,unfolded protein response,lipoprotein metabolism and lysosome biogenesis.