Date of Award

8-2011

Level of Access

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Anja Nohe

Second Committee Member

Robert Gundersen

Third Committee Member

Sharon Ashworth

Abstract

Bone morphogenetic protein 2 (BMP2) is a potent growth factor crucial for osteoblast differentiation and directs bone development and bone mineral density (BMD). BMP2 signals through two serine/threonine kinase receptors, BMP receptor type la (BMPRIa) and BMP receptor type Ia (BMPRIa), that are localized to distinct plasma membrane domains, clathrin coated pits (CCPs) and caveolae. The current BMP2 signaling model suggests the receptors localized to caveolae initiate Smad-independent pathways including p38 Map Kinase while receptor localization to CCPs activate the Smad-dependent pathway. Yet there are published data conflicting with this model. In order to define the role CCPs and caveolae have in BMP2 signaling the Family of Image Correlation Spectroscopy (FICS) was used to identify the regions at the plasma membrane BMPRIa was localized. The shuttling of BMPRIa in response to BMP2 was defined and was suggested to be involved in osteoblast differentiation and affected BMD. Caveolae were identified to be centers where BMP2 interacts with its receptors and regulates the Smad-dependent pathway. Further utilizing the collected receptor localization results by FICS and Smad-dependent signaling data from reporter gene assays, a mathematical model was derived which suggested caveolae contributed 78% of the Smad-dependent signal. In addition Adaptor protein 2 (AP2) and Casein Kinase II (CK2) were identified to be novel interacting proteins with BMPRII or BMPRIa and are thought to regulate the shuttling of the BMPRs at the plasma membrane. The interaction between CK2 and BMPRIa led to the development of blocking peptides which induced the Smad-dependent signal in the absence of BMP2. Injection of the peptide into an in vivo model led to increased bone growth. These results indicated BMP2 signaling through BMPRIa and BMPRII was highly regulated at the plasma membrane by both CCPs and caveolae. The shuttling of these receptors between these distinct membrane domains are needed for BMP2 signaling driving osteoblast differentiation, mineralization, and BMD.

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