Open AccessSplit‐receptors in the tachykinin neurokinin‐1 system
Author(s) -
Nielsen Soren Møller,
Elling Christian E.,
Schwartz Thue W.
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2510217.x
Subject(s) - substance p , receptor , intracellular , extracellular , tachykinin receptor , neurokinin a , tachykinin receptor 1 , inositol phosphate , chemistry , binding site , biology , biochemistry , biophysics , inositol , neuropeptide
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 (NK 1 ) 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 EC 50 for substance P and an E max according to the expression level. When intracellular loop 3 was either extended with 112 amino acid residues derived from the muscarine M 2 receptor or, when major parts of the loop were deleted in the non‐split NK 1 receptor, the affinity for neither substance P nor for the prototype non‐peptide antagonist, CP96,345 was affected, yet an increase in EC 50 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 NK, 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.