Luteinizing hormone receptors translocate to plasma membrane microdomains after binding of human chorionic gonadotropin.

Receptor-mediated signal transduction by G protein-coupled receptors can involve redistribution of plasma membrane receptors into membrane structures that are characterized by insolubility in Triton X-100 and low buoyant density in sucrose gradients. Here we describe the translocation of wild-type (wt) rat LH receptors (LHR-wt) from the bulk membrane into membrane microdomains (rafts) after the binding of human chorionic gonadotropin (hCG). In sucrose gradient ultracentrifugation of plasma membranes from cells stably expressing FLAG-tagged LHR-wt, receptors were located in high-density membrane fractions before binding of hormone and in low-density fractions after hCG treatment. Receptor translocation to low-density sucrose fractions did not occur when cells were pretreated with 1% methyl-beta-cyclodextrin, which reduces membrane cholesterol and disrupts rafts. Single-particle tracking of individual FLAG-LHR-wt receptors showed that hCG-treated receptors become confined in small compartments with a diameter of 86 +/- 36 nm, significantly smaller than 230 +/- 79 nm diameter regions accessed by the untreated receptor. Receptors were no longer confined in these small compartments after disruption of rafts by methyl-beta-cyclodextrin, a treatment that also decreased levels of cAMP in response to hCG. Finally, translocation of LHR into rafts required a functional hormone-receptor complex but did not occur after extensive receptor cross-linking that elevated cAMP levels. Thus, retention of LHR in rafts or small membrane compartments is a characteristic of functional, hormone-occupied LHR-wt. Although raft translocation was not essential for cAMP production, it may be necessary for optimizing hormone-mediated signaling.