Solving the Topology of DSPP’s Endoplasmic Reticulum Exit Cargo Receptor

Objectives: Dentin sialophosphoprotein (DSPP) is an acidic, secreted protein of the SIBLING protein family that is cleaved by BMP1 to give rise to dentin phosphoprotein (DPP) and dentin sialoprotein (DSP). DSP and DPP assemble into the extracellular matrix of dentin and are believed to promote dentin mineralization. The precursor protein, DSPP, starts with an amino-terminal Isoleucine-Proline-Valine (IPV) tripeptide. Many patients with nonsyndromic dentinogenesis imperfecta have mutations in their IPV-motif resulting in formation of DSPP aggregates in the endoplasmic reticulum (ER). We have proposed that a cargo receptor, CR1, binds DSPP through this IPV-motif and aids in trafficking the protein out of the ER before it builds to sufficient concentrations to form the aggregates. An IPV-like motif is also found in other acidic, secreted proteins and is highly conserved across species. The aim of the present study is to determine the number of transmembrane domains in CR1. ER cargo receptors are single or multipass transmembrane proteins with luminal domain(s) that bind soluble cargo proteins and cytosolic domain(s) that interact with COPII exit vesicle assembly proteins for trafficking to the Golgi.
Methods: We made antibodies against short peptides within several domains of CR1 and used different detergents to selectively open the plasma or the ER membranes of CR1-transfected HEK293 cells. Analysis of CR1 peptide domains was done by immunofluorescence microscopy.
Results: Based on different transmembrane domain prediction software, CR-1 is a multipass protein, but the number of transmembrane domains is unresolved. We have shown that the carboxyl terminus of CR1 resides on the cytosolic side of the ER membrane. Preliminary data are conflicting regarding the location of the amino terminus.
Conclusions: With continued study, we will determine the number of transmembrane domains of CR1 and determine its topology. This research was supported by the Intramural Research Program of the NIH, NIDCR.