Constitutive activation of chimeric m2/m5 muscarinic receptors and delineation of G-protein coupling selectivity domains

To derive structure/function relationships for muscarinic receptor/G-protein coupling, the m2 and m5 muscarinic receptors and a series of m2/m5 chimeras were tested for agonist binding and functional responses in a cellular proliferation/transformation assay. m5, which mediates stimulation of phosphatidylinositol turnover, displayed robust activity in the proliferation assay, whereas m2, which mediates inhibition of adenylyl cyclase, was inactive in the proliferation assay. Chimeras that contained m2 sequences in the i2 or i3 loops had impaired activity or were inactive, respectively. Chimeras that contained m2 segments reaching from the N-terminus to TM2, or from TM6 to the C-terminus, had enhanced activity relative to m5, and a chimera with both of these elements was constitutively activated.