Localization of luteinizing hormone receptors in small membrane compartments during signal transduction

Abstract

Luteinizing hormone receptors (LHR) are G protein-coupled membrane proteins with important functions in reproduction. These receptors, upon binding of hCG, translocate into plasma membrane compartments of low buoyant density. To further explore ligand-receptor structures necessary for localization of LHR in small membrane compartments, we have used single particle tracking methods to monitor the lateral diffusion of individual LH receptors and the size of plasma membrane compartments accessed by the receptor on viable cells. Following treatment of CHO cells expressing FLAG-tagged rat LHR with 100nM hCG, LHR become confined in compartments with an average diameter of 86 ± 36 nm. These compartments are significantly smaller than the 230 ± 79 nm diameter regions occupied by the untreated receptor. Following treatment of cells with 0.01nM hCG, LHR are distributed in two groups. Approximately 65% of the receptors are confined in small compartments with a diameter of 69 ± 38 nm while the remaining receptors exhibit unconfined lateral diffusion in large compartments typical of untreated LHR. Palmitoylation of the LHR C-terminus appears to be important for raft localization and for confinement in small membrane compartments. LHR mutants lacking potential palmitoylation sites at position 621 and 622 in the C-terminus do not translocate into membrane rafts. These receptors remain in large compartments and their rapid diffusion coefficients are not affected by hCG treatment. The cytoskeleton also appears to play a role in restricting hormone-treated receptors within small compartments: LHR on cells treated with cytochalasin D, a microfilament disruptor, exhibit fast lateral diffusion within large compartments both before and after exposure to 100nM hCG. Possible membrane models to explain this behavior include compartmentalization by cytoskeletally-anchored proteins that serve as protein fences. However, any such model must also explain why only hormone-occupied palmitoylated receptors are laterally restricted.