CRIM1 Regulates the Rate of Processing and Delivery of Bone Morphogenetic Proteins to the Cell Surface

Abstract

The Crim1 gene is predicted to encode a transmembrane protein containing six von Willebrand-like cysteine-rich repeats (CRRs) similar to those in the BMPbinding antagonist Chordin (Chrd). In this study, we verify that CRIM1 is a glycosylated, Type I transmembrane protein and demonstrate that the extracellular CRR-containing domain can also be secreted, presumably via processing at the membrane. We have previously demonstrated Crim1 expression at sites consistent with an interaction with bone morphogenetic proteins (BMPs). Here we show that CRIM1 can interact with both BMP4 and BMP7 via the CRR-containing portion of the protein and in so doing acts as an antagonist in three ways. CRIM1 binding of BMP4 and -7 occurs when these proteins are co-expressed within the Golgi compartment of the cell and leads to (i) a reduction in the production and processing of preprotein to mature BMP, (ii) tethering of pre-BMP to the cell surface, and (iii) an effective reduction in the secretion of mature BMP. Functional antagonism was verified by examining the effect of coexpression of CRIM1 and BMP4 on metanephric explant culture. The presence of CRIM1 reduced the effective BMP4 concentration of the media, thereby acting as a BMP4 antagonist. Hence, CRIM1 modulates BMP activity by affecting its processing and delivery to the cell surface.The TGF␤ 1 superfamily of ligands have prominent roles in nearly all tissues during development. There are over 30 members of this superfamily, including the TGF␤ isoforms, bone morphogenetic proteins (BMPs), and the closely related growth and differentiation factors, activins, and nodals (reviewed in Ref. 1). BMPs, like all members of the TGF␤ superfamily, are synthesized as preproteins, which are subsequently cleaved by furin and related subtilisin-like convertases within the Golgi apparatus at a conserved multibasic amino acid motif (Arg-X-X-Arg) to yield a dimeric C-terminal mature domain (2). Once secreted BMPs bind and facilitate the formation of heteromeric complexes of type I and type II serine/threonine kinase receptors (1). These signal via phosphorylation of Smad proteins, which can translocate into the nucleus and effect gene regulation and expression (reviewed in Ref. 1). Although TGF␤-1, -2, and -3 are secreted as a member of a large inactive complex containing the latency-associated peptide and a latent TGF␤-binding protein, the BMPs are currently thought to be processed and secreted as active mature molecules and not complexed with a latent TGF␤-binding protein-like molecule. However, like the TGF␤s, BMPs do not seem to act over a range of more than one to two cells, suggesting that there is some mechanism of "tethering" (3). This limit in signaling range is in part influenced by the prodomain of BMPs (3). The prodomain of the BMPs also influences the efficiency of processing and the stability of the mature BMP (2, 4). During development, TGF␤ superfamily members, and in particular BMPs, specify cell types by forming precisely controlled concentration gradients. The amount of activ...

Crim1KST264/KST264 mice display a disruption of the Crim1 gene resulting in perinatal lethality with defects in multiple organ systems

“…Importantly, it has been shown that the growth factor-binding ability of Crim1 largely resides in the CRR domains. While a deletion mutant lacking the region encoded by exon 2 but retaining the IG-FBP and CRR domains was capable of binding BMPs (Wilkinson et al, 2003), we would predict that the protein present in Crim1 KST264/KST264 mice is too short to contain a significant complement of CRR domains. As such, it may behave as an effective null.…”

“…One such protein is Crim1. We have previously shown that this transmembrane protein, which also contains an insulin-like growth factor binding protein (IG-FBP) motif and six vWF-C type cysteine-rich repeats, can modulate the activity of BMPs in a cell-autonomous manner in vitro (Wilkinson et al, 2003). In this study, we have characterized the phenotype of the Crim1 KST264 gene-trap mouse strain and detailed new sites of Crim1 expression based on a ␤-Geo reporter gene.…”

“…We have previously proposed that Crim1 may act as a modulator of the action of members of the TGF-␤ superfamily by means of the presence of CRR motifs Kolle et al, 2000;Wilkinson et al, 2003). Indeed, Crim1 can bind to BMP-2, -4, and -7 within the context of the cell, but not in solution (Wilkinson et al, 2003).…”

“…To determine whether there was a change in Crim1 protein levels in the mutant mice, we performed immunoblotting on the membrane fraction of 17.5 dpc wild-type, Crim1 ϩ/KST264 , and Crim1 KST264/KST264 whole kidney extracts. Using an antibody that detects the C-terminal, cytoplasmic portion of Crim1 (Wilkinson et al, 2003), we detected a predominant band of almost 150 kDa in wild-type samples Expression is also seen in the ovarian surface epithelium (BB, CC). da, dorsal aorta; ep, epicardium; lp, lamina propria; mu, muscularis; my, myocardium; nt, neural tube; pc, pericardium; te, transitional epithelium; tr, trabeculated myocardium.…”

“…We have demonstrated that Crim1 can bind to bone morphogenetic protein (BMP) -2, -4 and -7, but only within the context of the cell and not in solution, suggesting a cell-autonomous mode of action (Wilkinson et al, 2003). Crim1 forms a complex with the preprotein forms of such ligands, tethering the preproteins to the cell surface, thus retarding their secretion as mature active dimers (Wilkinson et al, 2003).…”