Structural correlates for down-regulation of m1 and m2 muscarinic receptor subtypes.

Three chimeric receptors stably expressed in murine fibroblast (B82) cells were used to examine how different parts of the rat muscarinic m1 and m2 receptors contribute to the down-regulation process. The MCH7 chimeric m2 receptor contained a fragment between VIth TM and C-terminal end derived from the m1 receptor. The MCH3 and MCH5 receptors have exchanged N-terminal and third intracellular loop regions of the MCH7 receptor. Fibroblast cells stably expressing individual muscarinic wild type (m1, 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 3H1-N-methyl-3-quinuclidinyl benzilate (3HMQNB) saturation binding studies. There was a significant loss of receptor density, different for each receptor studied, following carbachol treatment relative to control cells. We related this loss of 3HMQNB binding to the number of amino acids derived from m1 or m2 receptors for each constructed chimera and to the affinity of carbachol to the receptors studied. We demonstrate that: 1) the 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 and the interaction between intracellular portions of the receptor influence the extent of receptor down-regulation; and 3) resistance to down-regulation by carbachol correlates with the affinity of carbachol to the muscarinic receptor construct.