This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Full title: Investigation of roles of heparan sulfate proteoglycans (HSPGs) in bone morphogenetic protein-2 (BMP-2) ligand-receptor assembly and receptor oligomerization by fluorescence confocal microscopyHeparan sulfate proteoglycans (HSPGs) are highly glycated and sulfated proteins on cell surfaces and in the extracellular matrix. The linear and highly sulfated polysaccharide HS chains have been known to participate in signaling of several classes of polypeptide growth factors, including bone morphogenetic proteins (BMPs). It is known that BMPs involve in fundamental developmental processes as diverse as gastrulation, neurogenesis, chondrogenesis, dorsal-ventral patterning, and apoptosis. Exactly how HSPGs regulate BMP signaling remains unclear. Here we will investigate the roles of HS in BMP type II receptor pre-assembly, and BMP2 ligand-induced receptor oligomerization on cultured mammalian cell surface by employing FCM. Our preliminary biochemical data provided new insights into the mechanisms of HS regulating BMP actions, but did not allow us to look into higher-order receptor oligomerization in real time. Besides, HSPGs consist of huge amount of polysaccharide chains, and make it very difficult to apply some biochemical modification during experimental procedures. FCM will allow us to investigate the possible interactions among HSPGs, BMP ligand, and two types of BMP receptors without the limitations of biochemical experimental procedures. The following is what we plan to do:1. Detecting type II BMP receptor (BMPRII) dimerization upon enzymatic removal of HSPGs;2. Detecting possible association between BMPRII and glypican-1 (a predominant form of HSPGs in mammalian cells) upon BMP ligand stimulation; 3. Detecting oligomerization status of type IA BMP receptor (BMPRIA) and BMPRII upon BMP stimulation and/or removal of HSPGs.Currently we have mouse PC12 cells stably expressing BMPRII-EYFP (yellow fluorescence) and have successfully tested it before. We also have BMPRIA and glypican1 (both with fluorescence protein tags) constructs ready to use.
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