Organization of receptors for luteinizing hormone and gonadotropin releasing hormone during signal transduction

Abstract

Mechanisms involved in signal transduction by the luteinizing hormone (LH) and gonadotropin releasing hormone (GnRH) receptors are of considerable interest because of their importance in mammalian reproduction. Studying the organization of these receptors in the plasma membrane during signaling provides insight into protein interactions during a signaling response. This organization can be probed using biophysical methods. Using fluorescence energy transfer and fluorescence photobleaching recovery techniques, we have shown that LH receptors are found self-associated within slowly diffusing complexes whose size depends on whether LH or human chorionic gonadotropin (hCG) occupies the receptor. We have also created an intrinsically-fluorescent and functional LH receptor and measured its lateral diffusion in the absence and presence of ligand. Prior to binding of ligand the fluorescent receptor existed as a membrane protein whose diffusion coefficient was about 16 ± 3.5 x 10-10cm2sec-1. Upon binding of either LH or hCG, the rate of receptor lateral diffusion slowed and the fraction of mobile receptors decreased. Protein complexes containing the LH receptor formed in response to ligand binding were stable in the membrane while the receptor was desensitized. Non-responsive LH receptors remained self-associated and present in protein complexes. When receptors were again hormone-responsive, they exhibited fast lateral diffusion and were diffusely distributed in the membrane. Thus complexes that form in response to hormone binding must dissociate before the receptor can again respond to ligand. We also examined whether another G protein-coupled receptor, the GnRH receptor, self-associated in response to ligand binding. Lateral diffusion of an intrinsically-fluorescent GnRH receptor depended on the concentration of the applied ligand. However, only upon the addition of an agonist did we see a dose-dependent decrease in fraction of mobile receptors which was accompanied by an increase in the energy transfer efficiency between agonist-occupied GnRH receptors. From these data we have postulated a mechanism for LH receptor and GnRH receptor activation as well as a mechanism for LH receptor desensitization, each which involve the formation of protein complexes containing receptors that are self-associated.