Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662965
Title: Cloning and expression studies of the human type I inositol 1,4,5-trisphosphate receptor
Author: Ting-Kuang, N.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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Abstract:
In this study, a full-length human IP3RI was assembled from three overlapping fragments obtained from a human brain cDNA library. In initial work using in vitro coupled transcription/translation and in vivo expression in HEK293 cells, a C-terminally truncated IP3RI was obtained. Further sequence analysis identified a nucleotide deletion at position 6429, which caused the reading frame to be shifted and a stop codon to be generated at position 6455 resulting in a 200 kDa protein rather than 260 kDa. After correcting this by subcloning, full-length recombinant human IP3RI was expressed from an in vitro system and in HEK293 cells. Indirect immunofluorescence confirmed that overexpressed IP3RI was located in the ER. In contrast, the FL506-binding protein FKBP12, expressed from another cloned cDNA in the same cells, was mainly cytoplasmic. However, the overexpressed IP3RI did not have measurable IP3 binding activity, or channel activity when reconstituted in planar lipid bilayers, probably because of the low expression level following transient expression. Attempts to establish a stable cell line were unsuccessful, and a baculovirus expression vector system was therefore used to obtain high level expression. Full-length expressed IP3RI had an IP3 binding activity of about 0.6 pmol/mg of microsomes, which is much lower than that reported elsewhere. This suggests that further optimisation of expression is required. However, the truncated protein, which contained the N-terminal part of IP3RI responsible for binding IP3, bound about 3 pmol/mg of microsomes. This is similar to results published for other IP3Rs. Moreover, despite the absence of the C-terminus containing all the putative membrane-spanning domains, the truncated protein still appeared to be an intrinsic membrane protein. This suggests that there may be other membrane spanning domains in the human IP3RI not previously described in other IP3Rs. FKBP12 has previously been shown to associate with IP3RI. In future studies, coexpression of FKBP12 and IP3RI will be useful to study how FKBP12 modifies the function of recombinant human channels.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.662965  DOI: Not available
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