Jones, Caroline Jane; (2000) Cloning and regulation of the rat bradykinin B1 receptor gene. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Responses to vasoactive peptides are mediated by the action of two receptors, bradykinin B1 (BKB1) and bradykinin B2 (BKB2), which belong to the superfamily of G-protein coupled receptors. A role for inducible BKB1 receptors in pathophysiology has been demonstrated using pharmacological studies in a number of models of tissue injury and in response to specific inducing agents. This study describes the cloning of the rat BKB1 receptor gene and investigation of the molecular mechanisms involved in the inducible receptor expression. The first part of this thesis describes the isolation and characterisation of genomic and cDNA clones for the rat BKB1 receptor. This has allowed detailed analysis of the receptor protein coding sequence and determination of the BKB1 receptor genomic structure. Primer extension analysis and RACE studies have been used to identify the transcription initiation sites and the 3' cDNA ends and provide evidence for alternative splicing of the BKB1 receptor transcript. Furthermore, using the rat BKB1 receptor as a species-specific probe Northern blot analysis has established a distinct expression profile for the receptor mRNA in rat tissues and cultured cell lines. BKB1 receptor expression has been examined by Northern blot analysis in JTC-19 cells following the addition of a number of inducing agents. The increase in BKB1 receptor mRNA levels following the addition of the pro-inflammatory cytokine, IL-1β, and the bacterial endotoxin, LPS, were found to result from an increase in transcription with no change in mRNA stability. Examination of the effects of specifc inhibitors show that multiple signal transduction pathways, including PI 3-kinase activation and NF-κB-dependent and independent pathways are involved in the IL-1β and LPS-mediated BKB1 receptor transcriptional activation. Reporter gene constructs and electromobility shift assays have been used to delineate important elements involved in the transcriptional regulation of the BKB1 receptor gene.

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