Determining the Affinity of Hormone−Receptor Interaction

Abstract

Characterization of the binding of a hormone to its cognate receptor is a cornerstone of many studies in molecular and cellular endocrinology since this event represents the beginning of a specific cellular response, generally from a highly regulated extracellular messenger. The premise of hormone-receptor interaction follows from the law of mass action describing a reversible second-order reaction, hormone plus receptor, to give a non-covalently associated hormone-receptor complex. From this basic principle, a host of useful experimental parameters are available to the interested investigator. This chapter is focused on development of the experimental and mathematical underpinning of hormone-receptor interaction, with emphasis on a gonadotropin, chorionic gonadotropin (or luteinizing hormone), binding to the luteinizing hormone receptor, a member of the G protein-coupled receptor family. The general concepts and approaches developed herein are, however, valid to most interacting systems.

Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

“…In studies between proteins interacting with high affinity, such as FSH or hCG binding to the FSHR or LHR ECD, respectively, it is not surprising that disruption of single hydrogen bonds, ion-ion interactions, ion-dipole interactions, or even single hydrophobic interactions by Ala replacement fails to lead to a significant reduction in affinity. (Note that the binding assay used is not perfectly reversible because some hormone-receptor internalization can occur and some of the 125 I-labeled hormone may be degraded over the time course of the experiment (41). The K d values obtained, however, agree with measurements from numerous other laboratories, and thus confidence can be placed in the validity of the assay, at least when used for comparative purposes, as was done herein.)…”

“…Hormone Binding-Binding assays with HEK 293 cells were performed as given elsewhere (37,38,40,41 (42). Nonspecific binding was determined by addition of a 1000-fold excess of unlabeled hormone.…”

Conserved amino acids participate in the structure networks deputed to intramolecular communication in the lutropin receptor

“…Both competitive and saturation binding were done to determine the maximum receptor density (B max ) and either IC 50 (competition binding) or K d (saturation binding) [26]. K d was estimated from the IC 50 values using the relationship, K d = IC 50 -L, where L is the concentration of radiolabeled hormone.…”