SPLIT-RECEPTORS IN THE TACHYKININ NEUROKININ-1 SYSTEM - MUTATIONAL ANALYSIS OF INTRACELLULAR LOOP-3

Several membrane proteins have been functionally expressed from non-co valently coupled, contiguous segments especially with the split-site l ocated between natural domains. Experiments using such 'split-proteins ' were here performed in the tachykinin neurokinin-1 (NK1) receptor wi th co-expression of contiguous segments with split-sites positioned in various intracellular and extracellular loops. The construct where th e split-site was located in intracellular loop 3 gave a reasonable exp ression level of substance-P-binding sites, i.e. 12% of wild-type expr ession. Of the other split-receptors tested, only the one with the spl it-site located just outside transmembrane (TM) segment-V gave any det ectable substance P binding, which however only was 1% of the wild-typ e expression level. The construct with the split-site located in intra cellular loop 3 bound all of the tested peptide agonists and non-pepti de antagonists with normal affinity and was able to stimulate inositol phosphate turnover with a normal EC50 for substance P and an E-max ac cording to the expression level. When intracellular loop 3 was either extended with 112 amino acid residues derived from the muscarine M-2 r eceptor or, when major parts of the loop were deleted in the non-split NK1 receptor, the affinity for neither substance P nor for the protot ype nonpeptide antagonist, CP96,345 was affected, yet an increase in E C50 for substance P was observed. Also in the split-receptor, most of intracellular loop 3 could be substituted or even deleted without affe cting ligand affinity, although a decreased expression level was obser ved in constructs having major deletions. It is concluded, that the NK , receptor is preferentially reconstituted by co-expression of a putat ive A-domain including TM-I-V and a B-domain including TM-VI and -VII. It is suggested that a number of rhodopsin-like 7TM receptors may fun ction as two-domain structures based on the finding that a network of short loops has been highly conserved within each of the putative doma ins and, that these domains are separated by a relatively long and in respect of length poorly conserved loop, i.e. intracellular loop 3.