Ligand binding kinetics of substance P and neurokinin A receptors stably expressed in Chinese hamster ovary cells and evidence for differential stimulation of inositol 1,4,5-trisphosphate and cyclic AMP second messenger responses.
Stably transfected Chinese hamster ovary cells expressing either the substance P receptor or neurokinin A receptor were constructed, isolated, and characterized. Equilibrium ligand binding studies performed on whole cells demonstrated that cell lines expressing either of these receptors contained a single class of high-affinity binding sites with an apparent KD of 0.16 nM for the substance P receptor and an apparent KD of 2.1 nM for the neurokinin A receptor. The higher affinity of substance P for its receptor was accounted for by both a greater association rate constant and a lesser dissociation rate constant. The time course and extent of ligand-stimulated inositol 1,4,5-trisphosphate mass increases in both cell lines were similar and displayed rapid and transient kinetics. Ligand-stimulated cyclic AMP accumulation was also apparent in the cell lines, although the time course and magnitude of the responses were substantially different, with the neurokinin A receptor mediating a greater and more prolonged response. These studies establish the presence of functional substance P receptors and neurokinin A receptors in the stably transfected cell lines and provide evidence for agonist-dependent differential stimulation of second messenger responses.
Takeda
Y
Y
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Blount
P
P
Sachais
B S
BS
Hershey
A D
AD
Raddatz
R
R
Krause
J E
JE
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism* / Q000502:physiology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D018040:Receptors, Neurokinin-1; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D015290:Second Messenger Systems / Q000502:physiology*
D008024:Ligands; D018040:Receptors, Neurokinin-1; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP