The Interactions for the RBP Receptor, Stimulated by Retinoic Acid Gene 6

Vitamin A, or retinol, is a fat-soluble vitamin, essential for many important processes including proliferation, reproduction, vision and immunocompetence. Retinol binding protein (RBP) is the principal carrier of retinol in the blood from its storage site, the liver, to retinol dependant tissues. The long sought RBP receptor was recently identified as stimulated by retinoic acid gene 6 (STRA6), a 74 kDa multitransmembrane protein with no similarity to any other protein receptor. At present, receptor function, architecture and structure-function relationships remain relatively unexplored. Identification of any protein(s) that interact with STRA6 is imperative in identifying any other role STRA6 may play in the body. The membrane yeast two hybrid (MYTH) represents a powerful tool to facilitate the characterisation of membrane protein interactions and was utilized to detect proteins that interact with full-length STRA6. A human kidney and a human brain library were screened. The human kidney screen was repeated in the presence of RBP-ROH to identify any protein interactions dependent on the presence of holo-RBP. The MYTH screens resulted in 11 unique protein interactions for STRA6. These interactions could not be verified using pull-down experiments despite numerous attempts. In the hope of gaining insights into STRA6’s architecture and function, the predicted large third intracellular loop (ICL3) of STRA6 was extracted from the full length protein, introduced into a carrier protein, Im7, and characterised. STRA6-ICL3 was found to form a tetramer. The native Im7 is monomeric; therefore, the domain conferred tetrameric behaviour. These data suggest that native STRA6 is likely to occur as a tetramer and that at least some of the oligomerization sites are located on this large intracellular loop. By determining the circular dichroism spectra for STRA6-ICL3, the first structural data for STRA6 was collected. These data revealed that STRA6-ICL3 was mostly composed of β-sheet and random coil. Crystallization trials were also attempted and a crystal is eagerly awaited.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

National University of Ireland Maynooth

Year dc:date.issued

2013

Author dc:creator

• McQuaid, Kate

• Biology

Language dc:language

en