FROM PROTEIN TO SIGNALPLEX: THE ROLE OF CALCIUM BINDING AND MYRISTOYLATION IN NEURONAL CALCIUM SENSOR-1 STRUCTURE AND FUNCTION
Open Access
- Author:
- De Cotiis, Daniel A
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 02, 2009
- Committee Members:
- John Michael Flanagan Jr., Dissertation Advisor/Co-Advisor
John Michael Flanagan Jr., Committee Chair/Co-Chair
Maria Christine Bewley, Committee Member
Robert G Levenson, Committee Member
Thomas E Spratt, Committee Member
Ira Joseph Ropson, Committee Member - Keywords:
- neuronal calcium sensor 1
signalplex
myristoylation
dopaminergic signalling - Abstract:
- Neuronal Calcium Sensor 1 (NCS-1) is an acidic, highly helical protein (MW 23 kDa), which is involved in a variety of cellular pathways including exocytosis and G-protein linked receptor desensitization. NCS-1 contains an N-terminal myristoylation modification, as well as three EF-hand domains which bind calcium with moderate to high affinity. These attributes have been previously shown to affect the structural and functional behavior of the protein. In order to better characterize these changes, a protocol presented here has been designed and optimized to produce large quantities of recombinant N-myristoylated and unmodified NCS-1 in E. coli. Using this technique, novel mutants showing a spectrum of functional deficiencies are expressed, isolated and then characterized by a number of assays. The large quantities of homogenous protein produced are shown to be suitable for biophysical studies using a variety of in vivo and in vitro techniques. Contrary to what would be expected for a protein of small size, NCS-1 interacts with a wide diversity of other proteins. One mechanism by which NCS-1 could assume this functional assortment of roles is by forming higher order homo-complexes. Experimental results presented here demonstrate that NCS-1 is capable of forming calcium dependent homo-dimer complexes. The propensity for self-association is enhanced by N-terminal myristoylation. In the absence of ligand, all three functional EF-hand sites must be intact in order for NCS-1 dimerization to occur. As NCS-1 plays a wide diversity of roles in the cell, the formation of homo-dimers may have pronounced functional implications. NCS-1 forms a signaling complex with the D2 dopamine receptor (D2R) and other downstream effectors, ultimately regulating desensitization of the receptor in response to dopamine signaling. As a result, this regulatory complex may be involved in a number of neurological disease states including schizophrenia and bipolar disorder. A better understanding of the biophysical and regulatory details of this complex will have important implication for understanding disease pathology and exploring novel drug treatments. Using a fluorescent peptide substrate in a fluorescence polarization assay, the equilibrium binding behavior of the NCS-1 / D2R interaction is described here. The formation of the NCS-1 / D2R complex is shown to be calcium dependent and involve the NCS-1 dimer. The contribution of the various EF-hand domains to binding is also investigated, and a discrete role in dimerization and binding is implied for each pair of EF-hand domains. The outcomes of these experiments could be used to optimize a large format drug screen scaled from this assay system, which could ultimately isolate lead compounds for the treatment of D2R related diseases.