Structural correlates for down-regulation of m1 and m2 muscarinic receptorsubtypes

Three chimeric receptors stably expressed in murine fibroblast (B82) cells were used to examine how different parts of the rat muscarinic m1 and m2 re ceptors contribute to the down-regulation process, The MCH7 chimeric m2 rec eptor contained a fragment between VIth TM and C-terminal end derived from the mi receptor, The MCH3 and MCH5 receptors have exchanged N-terminal and third intracellular loop regions of the MCH7 receptor, Fibroblast cells sta bly expressing individual muscarinic wild type (ml, m2) or chimeric (MCH3, MCH5, or MCH7) receptors were treated with plain medium (control) or medium containing carbachol for 24 h, Receptor density changes were measured by [ H-3](-)1-N-methyl-3-quinuclidinyl benzilate ([H-3](-)MQNB) saturation bindi ng studies. There was a significant loss of receptor density, different for each receptor studied, following carbachol treatment relative to control c ells. We related this loss of [H-3](-)MQNB binding to the number of amino a cids derived from mi or m2 receptors for each constructed chimera and to th e affinity of carbachol to the receptors studied. We demonstrate that: 1) t he region from the VIth TMD to the end of C-terminal controls the extent of m1 and m2 receptor down-regulation; 2) the overall receptor conformation a nd the interaction between intracellular portions of the receptor influence the extent of receptor down-regulation; and 3) resistance to down-regulati on by carbachol correlates with the affinity of carbachol to the muscarinic receptor construct. (C) 1998 Elsevier Science Inc.