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Title: Biophysical characterisation of the Cannabinoid Receptor interacting protein CRIP1a
Author: Saul, Louise
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2012
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Endogenous cannabinoids, such as anandamide and 2-arachidonoyl-glycerol, are compounds naturally produced in the body that act as important signalling modulators, particularly, in the brain. Endocannabinoids bind to cannabinoid receptors (the CB1 and CB2 receptors), the same receptors to which the psychoactive tetrahydrocannabinol ingredient of Cannabis saliva derivatives bind. Endocannabinoids, together with their receptors, and the protein network involved in endocannabinoid synthesis, uptake, release and degradation constitute the endogenous cannabinoid system. The endogenous cannabinoid system is an emerging target in pharmacotherapy for the treatment of several diseases. In the context of a project aimed at studying important proteins of the ECS attention was focused on a 19kDa protein called Cannabinoid Receptor Interacting Protein la (CRIP1a). CRIP1a has been recently discovered, using a yeast-two-hybrid approach, as a binding partner of the CB1 receptor, but not the CB2 receptor. CRIP1a has been suggested to bind to the C-terminal cytoplasmatic tail of the CB1 receptor and to reduce the CB1 receptor-mediated tonic inhibition of voltage-gated calcium currents. The CRIP1a rat ortholog, RnCRIP1a, was over-expressed in E. coli and purified to homogeneity. Single crystals of wild-type and selenomethionine-labelled RnCRIP1a were grown using the microseeding technique and diffracted to high resolution using synchrotron radiation. The structure of RnCRlP1a was solved using the multiple anomalous diffraction technique at a resolution of 1.8 Å. RnCRIP1a is a domain-swapped dimer in the crystal. Its C-terminal region is in an extended β-strand conformation, 'crossing over' to engage in a β-sheet symmetry-related molecule, thus resulting in the formation of a homodimer. Analytical ultracentrifugation and NMR experiments show, however, that RnCRIP1a is a monomer in solution. On the basis of homonuclear NOE signals, in solution RnCRIP1a appears to have its C-terminus folded back into the β-sandwich. In consideration that RnCRIP1a C-terminal region harbours a PDZ-binding consensus sequence, the possibility of a functional role for the opening of its C-terminal is intriguing. The RnCRIP1a monomer displays a modified immunoglobulin fold equipped with a 'lid' domain above its β-sandwich scaffold. From a topological point of view, RnCRIP1a resembles members of the Ig-fold family of DNA binding proteins. RnCRIP1a has been proposed to bind to the last 55 amino acids of the CB1 receptor. This was tested by in-vitro pull-down and NMR titration experiments and failed any interaction between RnCRIP1a and the C-terminal tail of the CB1 receptor. Subcellular localisation assessed by confocal microscopy using COS7 cells reveals that RnCRIP1a is predominantly a cytoplasmatic protein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available