title: Split-receptors in the tachykinin neurokinin-1 system--mutational analysis of intracellular loop 3.
abstract: Several membrane proteins have been functionally expressed from non-covalently coupled, contiguous segments especially with the split-site located between natural domains. Experiments using such 'split-proteins' were here performed in the tachykinin neurokinin-1 (NK1) receptor with co-expression of contiguous segments with split-sites positioned in various intracellular and extracellular loops. The construct where the split-site was located in intracellular loop 3 gave a reasonable expression level of substance-P-binding sites, i.e. 12% of wild-type expression. Of the other split-receptors tested, only the one with the split-site located just outside transmembrane (TM) segment-V gave any detectable substance P binding, which however only was 1% of the wild-type expression level. The construct with the split-site located in intracellular loop 3 bound all of the tested peptide agonists and non-peptide antagonists with normal affinity and was able to stimulate inositol phosphate turnover with a normal EC50 for substance P and an Emax according to the expression level. When intracellular loop 3 was either extended with 112 amino acid residues derived from the muscarine M2 receptor or, when major parts of the loop were deleted in the non-split NK1 receptor, the affinity for neither substance P nor for the prototype nonpeptide antagonist, CP96,345 was affected, yet an increase in EC50 for substance P was observed. Also in the split-receptor, most of intracellular loop 3 could be substituted or even deleted without affecting ligand affinity, although a decreased expression level was observed in constructs having major deletions. It is concluded, that the NK1 receptor is preferentially reconstituted by co-expression of a putative 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 function as two-domain structures based on the finding that a network of short loops has been highly conserved within each of the putative domains and, that these domains are separated by a relatively long and in respect of length poorly conserved loop, i.e. intracellular loop 3.
authors.affiliation: Department of Pharmacology, Panum Institute, University of Copenhagen, Denmark.
authors.forename: S M
authors.initials: SM
authors.lastname: Nielsen
authors.forename: C E
authors.initials: CE
authors.lastname: Elling
authors.forename: T W
authors.initials: TW
authors.lastname: Schwartz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001713:Biphenyl Compounds / Q000378:metabolism; D019556:COS Cells / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010716:Phosphatidylinositols / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013373:Substance P / Q000378:metabolism
chemical_list: D001713:Biphenyl Compounds; D010716:Phosphatidylinositols; D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins; D013373:Substance P; D019204:GTP-Binding Proteins; C066892:CP 96345