Kinetic and biophysical analysis of the m2 muscarinic receptor.
The recombinant Pm2 muscarinic receptor expressed in Chinese hamster ovary (CHO) cells was used as a model system to examine receptor-effector coupling and ligand binding. In CHO cells, equilibrium binding studies and the dependence on receptor number per cell of the maximum response and EC50 values for agonist stimulation of phosphatidylinositol metabolism and inhibition of cAMP formation were consistent with a modified ternary complex model of signal transduction that included a physiologically noncompetent receptor state. Detailed kinetic studies of oxotremorine M (Oxo-M) binding to CHO cell membranes suggested that agonist interactions at the high affinity class of binding sites are complicated and depend on receptor expression levels. At low levels of expression, kinetic data were consistent with a special case of a mechanism in which Oxo-M shifts the equilibrium between two receptor conformations while at high levels of expression, it was necessary to evoke receptor-receptor interactions to explain the kinetic data. Far ultraviolet circular dichroism studies of the purified recombinant receptor showed a high content of alpha-helical secondary structure and small changes in secondary structure upon antagonist, but not agonist, binding.
Hirschberg
B T
BT
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-7305, USA.
Mosser
V A
VA
Peterson
G L
GL
Toumadje
A
A
Vogel
W K
WK
Johnson
W C
WC
Schimerlik
M I
MI
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D018721:Muscarinic Agonists / Q000378:metabolism; D010095:Oxotremorine / Q000031:analogs & derivatives / Q000378:metabolism; D017433:Protein Structure, Secondary; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D013056:Spectrophotometry, Ultraviolet; D014162:Transfection
D000067956:Adenylyl Cyclase Inhibitors; D008024:Ligands; D018721:Muscarinic Agonists; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D010095:Oxotremorine; C042743:oxotremorine M; D000242:Cyclic AMP; D000262:Adenylyl Cyclases