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PhD Thesis: Katherine Figueroa

Dissertation Abstract:
Measurement of GPCR agonist activity using Intrinsic Relative Activity

By Katherine W. Figueroa
Doctor of Philosophy in Pharmacology and Toxicology
University of California, Irvine, 2008
Professor Frederick J. Ehlert, Chair

An Intrinsic Relative Activity (RAi) value of agonist activity provides an estimate of its microscopic affinity constant for the active state of a G-protein-coupled receptor (GPCR)-G protein complex relative to that of a standard agonist. It can also be described as the product of intrinsic efficacy and observed affinity of an agonist relative to a standard.

Two methods are available for determining the RAi value of an agonist, the operational method and the null method. The former is based upon describing one response relative to another if they are both obtained in the same system and the concentration-response curves resulting from their activation of the receptor can be fit according to the ‘Operational Model’ described by Black and Leff in 1983. The second method is based upon equalization of two equipotent responses of two agonists obtained in the same system and fitting them to an algebraic equation providing a ratio of their intrinsic efficacy and observed affinity, which is equal to RAi. The RAi value of an agonist will remain the same between different functional assays if the GPCR-G protein complex transducing the initial stimulus is the same.

Chapter 2 examines the use of RAi in comparing agonist activity across functional assays based upon the 5-HT4 serotonergic receptor. It was used to determine if selected agonists can differentiate between the GPCR-G protein complexes expressed in the different assays tested.

Subsequently, a partial 5-HT4 receptor agonist, RS 67506, was shown to differentiate between the 5-HT4 receptor endogenous to the isolated rat tunica muscularis versus the rat 5-HT4(b) receptor transfected into HEK-T cells.

Chapter 3 investigates the use of RAi for highlighting a change in agonist activity in cases of alternative G protein signaling. R-aceclidine, a partial muscarinic agonist, was identified as having increased activity at the human M4 muscarinic receptor if it signals via Gα15 instead of its predominant signaling pathway utilizing Gi. Chapter 4 investigated the binding site of McN-A-343 at the human M1 muscarinic receptor by studying the change in receptor alkylation kinetics of nitrogen mustard derivatives of acetylcholine and McN-A-343. The pattern of inhibition by known orthosteric and allosteric muscarinic ligands suggests the binding site of McN-A-343 at the M1 receptor is orthosteric.