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Amino terminal protein sequencing; Human B2 receptor; Baculovirus expression system
Abstract:
Receptors for kinins are classified into B 1 (Menke et al., 1994) and B2 (McEachern et al., 1991) sub- types. Both receptors belong to the seven transmembrane domain (TMD) receptor family which activate trimeric G-proteins. The cDNAs coding for the rat (McEachern et al., 1991), the human (Hess et al., 1992; Eggerickx et al., 1992) and the mouse (Yokoyama et al., 1994) B2 receptors contain several in-frame methionine codons which could be used as an initiation site for translation; however, none of them conforms to the 'canonical' consensus sequence often preceding the initiation codon (Kozak, 1989). On the basis of sequence similarity studies the third in-frame AUG of the human B2 receptor mRNA, starting at nucleotide 223 of clone CCD-16- 2, was assumed to be the preferred start codon (Hess et al., 1992). The underlying gene of the human B2 receptor consists of three exons interrupted by two introns (Ma et al., 1994): exon-1 is non-coding, the first two in-frame AUG codons are located on exon-2, and the third in-frame AUG is located on exon-3 together with the seven transmembrane spanning sequence segments. There is no evidence for alternative splicing of the human B2 receptor pre-mRNA (Kammerer et al., 1995). To clarify which in-frame AUG serves as the translation initiation codon, we have isolated the B2 receptor protein and determined its amino-terminal sequence (Abd Alia et al., in preparation). The sequence data indicate that the B2 receptor mRNA from human foreskin fibroblasts is translated from the first in-frame initiator AUG codon. We report here that the extra segment of 27 amino acid residues which precedes the predicted amino-terminus of the receptor protein does not significantly alter the binding characteristics of the receptor.
