MONTAJE

This chapter describes the elucidation of the exon-intron structure of the rat BKBi receptor gene. This was achieved by isolation and characterisation of cDNA and genomic clones. Using these sequences as specific probes, a distinct expression pattern for the BKB] receptor gene in a number of tissues and the rat cell line, JTC-19, has been determined.

The rat BKB] receptor cDNA clone contains a single ORF encoding a G-protein coupled receptor of 338 amino acids, and is identical to that recently described by Ni and colleagues (1998b). The structure of the rat receptor shows a high degree of similarity to the mouse BKB] receptor (Hess et al, 1996; Pesquero et al, 1996). In contrast, both rodent receptors show marked differences with the human and rabbit receptor sequences. The large insertion of basic amino acids in the first intracellular loop together with the shortened C-terminal cytoplasmic tail, may be reflected in differences in coupling of the rat and human receptors to heterotrimeric G-proteins (Strader et al, 1994; Wong and Ross, 1994; Capeyrou et al, 1997). Significant divergence of the BKB] and BKB2 receptors has been noted previously (Menke et al,

1994). Despite their limited homology to each other, the BKBi and BKB2 receptors are

obviously functionally related since they are both activated by kinins. Indeed, the human receptor genes are located close together on human chromosome 14q32 (Ma et al, 1994a; Kammerer et al, 1995; Pesquero et al, 1994; Chai et al, 1996; Bachvarov et al, 1998a). The BKBi and BKB2 receptors are most likely derived by divergent

evolution from the same ancestral gene which may resemble the chicken omithokinin receptor (Schroeder et al, 1997). The structural differences between the BKB]

receptors from different species represents further divergent evolution within this subgroup of receptors.

Following the isolation of the rat BKBi receptor, the pharmacological properties of the rat and human receptors were examined by investigating the ability of a range of agonists and antagonists to inhibit the binding of [^H]desArg^\allidin (This work was carried out by other colleagues in the laboratory and is reported in Jones et al, 1999). These studies showed that the rat BKBi receptor has higher affinity for des- A rg^\allidin (Ki value 1.6 ± 0.2 nM) than desArg^BK (Ki value 15 ± 5 nM), although Ni et al, (1998b) report that these ligands have equal affinity for the rat receptor. Significantly, at the human receptor, desArg^\allidin was about ten times more potent than at the rat receptor (Ki value 0.19 ± 0.05 nM), with 5,000 fold greater affinity compared to desArg^BK (Ki value 1620 ± 153). In addition, the potential kininase I derivative of T-kinin, desArg'^T-kinin had relatively high affinity for the rat BKBi receptor (Ki value 46 ± 14 nM), with 30 fold lower activity at the human receptor. Therefore, it would appear that due to differences in the kinin systems between rodents and humans (see Chapter 1 section 1.1), the human receptor has evolved greater sensitivity towards kallidin and desArg^\allidin and the rodent receptor has evolved sensitivity towards bradykinin and T-kinin derivatives. Other studies have shown that the mouse BKBi receptors exhibit a 2 to 3 fold greater selectivity for desArg^BK over desArg^\allidin (Hess et al, 1996; Pesquero et al, 1996). The region of the receptor responsible for these binding affinities has not been elucidated. Whether these different pharmacological properties can be explained by differences between the species in the first intracellular loop and the C-terminus remains to be determined. Studies carried out

on chimeric BKB] and BKB2 receptor proteins have shown that the TM VI of the BK

receptor affects the binding of desArg^\allidin (Leeb et al, 1997). Alignment of the BKB] receptor protein sequences over TM VI and the third extracellular loop has shown the location of a number of amino acids that are different between the rodent receptors compared to the rabbit and human receptors (Jones et al, 1999).

The findings presented in this chapter show that the rat BKB] receptor gene structure consists of two exons, this differs from that seen in the human receptor gene which spans three exons (Bachvarov et al, 1996; Yang and Polgar, 1996). However the coding domain of both the rat and human receptors are located on a single exon. Preliminary observations of the human and rat BKB] receptor exon-intron sizes suggested that exon 1 of the rat is equivalent to exon 2 of the human BKBj receptor and that the rat BKB] receptor gene was incompletely characterised. However, alignment of the non-coding sequence presented here shows that exons 1 of the rat and human receptor genes are homologous indicating that the differences in gene structure are species-specific. The BKB] receptor genomic structure for other organisms remains to be determined. The data show the presence of two adjacent TISs. In contrast, a recent report by Ni and co-workers (1998b) describes the existence of a TIS located 20 bps upstream from the start sites reported in this study. Similar discrepancies have been reported for the identification of the TIS in the human BKB] receptor gene (Bachvarov

et al, 1996; Yang and Polgar, 1996). These data suggest that there may be subtle differences in the transcription of the gene in different cell types. No evidence was found from primer extension or 5’RACE for the existence of a TIS at the beginning of the coding exon, analagous to that suggested for the human BKB] receptor in lMR-90 cells (Yang and Polgar, 1996). Analysis of 5’RACE products shows the existence of an

alternative splice acceptor site, a finding recently reported by Ni et al, (1998b). The downstream splice acceptor site is equivalent to the exon-intron junction observed in the human BKBi receptor gene (Bachvarov et al, 1996; Yang and Polgar, 1996). However, it is not known if similar splice variants exist in the human BKBi receptor mRNA. Analysis of the expression of these splice variants is described in Chapter 4. Investigation of the 3’ cDNA ends of the rat BKBi receptor gene by 3’ RACE revealed the existence of alternate polyadenylation usage, although alternative transcripts generated by these sites were not detected by Northern blot analysis. However, it is possible that variations in polyadenylation of the transcripts generated by these sites render them indistinguishable by Northern blot studies. In addition, hybridisation experiments using a 3’ genomic fragment showed that the large mRNA transcripts of 4 and 8 kb are generated by alternative 3’ processing of the BKBi receptor gene.

The BKB] receptor gene expression is induced under some pathophysiological conditions. In particular, in vivo induction of the BKB] receptors has been characterised in the mediation of inflammatory and cardiovascular responses following treatment with a sub-lethal dose of LPS (Campos et al, 1996; Regoli et al, 1981; Siebeck et al, 1989, 1996, 1997; Tokumasu et al, 1995; Nicolau et al, 1996). The present study shows that the rat BKB] receptor mRNA is up-regulated by LPS in a distinct tissue expression pattern. In all the tissues tested the mRNA was up-regulated from basal levels. In uterus, bladder and lung the final levels of BKB] receptor mRNA were particularly high compared with the other tissues. Whether this increase in mRNA results in an increase in BKB] receptor protein has not been shown directly. However, it is well established for the majority of genes that the difference in mRNA expression levels are paralleled by differences in protein composition (Latchman, 1995). The basal levels of BKB]

receptor mRNA in uterus were 10 fold higher than in any of the other tissues tested. The significance of BKBi receptor mRNA under normal conditions remains unclear but one can speculate that the BKBi receptor is important in the function of the uterus and may be regulated by oestrogen as has been shown for the BKB2 receptor (Madeddu et

al, 1997b). However, another study showed high levels of receptor mRNA in the rat prostate with no effect of the sex hormones on BKBi receptor mRNA levels (Ni et al,

1998b). A separate study has shown a similar tissue expression pattern for BKBi receptor mRNA as that reported in this chapter, with additional tissues showing increased receptor mRNA in duodenum and undetectable levels of receptor mRNA in brain, spinal cord and DRG (Jones et al, 1999). Other studies have analysed the BKBi receptor mRNA levels in rats and mice by RT-PCR following LPS treatment (Pesquero

et al, 1996; Ni et al, 1998b; Marin-Castano et al, 1998). The induction of BKBi receptor mRNA resulted in increased functional receptor expression in the rat nephron (Marin-Castano et al, 1998). Thus, the BKBi receptor up-regulation produced by LPS in different in vitro and in vivo systems is caused by mRNA induction. However, within this up-regulation there is a great deal of heterogeneity, this could be due to a number of effects including tissue-specific regulation, the ability of the tissues to generate an inflammatory response or the local concentration of LPS between tissues.

Data presented in this chapter shows that the rat fibroblast cell line, JTC-19, and the mouse fibroblast cell line, NIH-3T3 increase BKBi receptor mRNA levels following treatment with IL-1(3 in a similar way to that observed with the human fibroblast cell lines, IMR-90 and WI38 (Bachvarov et al, 1996; Phagoo et al, 1997,1999; Zhou et al,

1998; Shanstra et al, 1998; B asti an et al, 1998). More recent studies have shown that rat VSMCs, and the rat cell line, A 10, express inducible BKBi receptor mRNA (Ni et

al, 1998a,b). BKBi receptor mRNA levels in AlO cells are induced 3-4 fold following the addition of inducing agents. However, the 16 fold induction observed following IL- Ip treatment in JTC-19 cells resembles the increases observed in rat tissues. In contrast, expression of BKBi receptor mRNA is non-inducible in rat hepatoma H4 cells.

In summary, these data show the isolation, characterisation and expression of the rat BKBi receptor gene. The identification of JTC-19 cells as a cellular model for analysis of BKB] receptor up-regulation will allow detailed analysis of the mechanisms of BKB] receptor gene expression.

Chapter 4