Previous results from this laboratory suggested that the same active conformation of the lutropin/choriogonadotropin receptor (LHR) is involved in the stimulation of G proteins and in triggering the internalization of the bound agonist.We have now analyzed two naturally occurring, constitutively active mutants of the human LHR. These mutations were introduced into the rat LHR (rLHR) and are designated L435R and D556Y. Cells expressing rLHR-D556Y bind human choriogonadotropin (hCG) with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation does not affect the internalization of the free receptor, but it enhances the internalization of the agonist-occupied receptors ϳ3-fold. Cells expressing rLHR-L435R also bind hCG with normal affinity, exhibit a 47-fold increase in basal cAMP, and do not respond to hCG with a further increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ϳ2-and ϳ17-fold, respectively. We conclude that the state of activation of the rLHR can modulate its basal and/or agonist-stimulated internalization.Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing rLHR-L435R is due to the fast rate of internalization of the bound hCG. The finding that membranes expressing rLHR-L435R (a system where internalization does not occur) respond to hCG with an increase in adenylyl cyclase activity supports this suggestion. The lutropin/choriogonadotropin receptor (LHR)1 is a member of the rhodopsin-like subfamily of G protein-coupled receptors (GPCRs) (reviewed in Ref. 1) that has been shown to mediate the internalization of its two naturally occurring agonists, lutropin and choriogonadotropin (CG). These studies, which have been conducted in target or transfected cells, have shown that the free LHR is randomly distributed in the plasma membrane, but that it clusters in clathrin-coated pits upon agonist activation (2). The clustered agonist-receptor complex is internalized by a dynamin-dependent pathway and traverses the endosomal compartment without agonist dissociation (3-5). Dissociation of the agonist-receptor complex occurs in the lysosomes, where both the agonist and the receptor are degraded (2, 4 -6). The receptor-mediated endocytosis of lutropin and CG serves two important functions in regulating cellular responsiveness. First, the internalization of the receptor-bound agonist effectively stops activation of downstream effectors such as adenylyl cyclase (7). Second, the accumulation of the agonistreceptor complex in the lysosomes promotes receptor degradation and is ultimately responsible for the agonist-induced down-regulation of the cell surface LHR, an outcome that prevents further activation of the cells (4,8,9).The lysosomal accumulation of the rLHR-agonist complex and the involvement of rLHR internalization in the termination of hormone action (i.e. desensitizat...

Previous studies from several laboratories have shown that the cell surface rLHR is a 85-92 kDa protein synthesized from a 68-73 kDa intracellular precursor. While all investigators agree that the cell surface rLHR binds hCG with high affinity, it is not clear if the intracellular precursor can also bind hCG. In order to directly determine if the intracellular rLHR present in cells transfected with the wild-type rLHR binds hCG with high affinity, we devised a method that selectively degrades the cell surface rLHR while preserving the intracellular rLHR. The binding of hCG to intact cells was completely lost following mild proteolysis of the cells, but binding to detergent extracts prepared from proteolyzed cells was largely preserved. Measurements of the hCG binding affinity to intact cells or to detergent extracts prepared before and after proteolysis display very similar or identical binding affinities. Since binding to nonproteolyzed intact cells, detergent extracts prepared from nonproteolyzed cells, or detergent extracts prepared from proteolyzed cells occurs only to the 85-92 kDa rLHR, the 85-92 and 68-73 kDa rLHR, and the 68-73 kDa rLHR, respectively, we conclude that the cell surface rLHR and the intracellular rLHR bind hCG with the same affinity. Quantitation of the relative abundance of the cell surface and intracellular rLHR by immunological methods indicates that transfected cells express mostly the intracellular precursor. A comparison of the binding capacity of control and proteolyzed cells with that of their detergent extracts indicates that hCG binding assays greatly underestimate the relative abundance of the intracellular rLHR.

The experiments presented herein were designed to study the molecular basis of the restricted cellular localization and transcriptional regulation of the LH/CG receptor in Leydig cells. Using luciferase fusion constructs transfected into Leydig and Sertoli cell lines, we show that the proximal 186 basepairs (relative to the translation start site) of the 5'-flanking region of the rat LH/CG receptor represent a basal promoter that accounts for the Leydig cell-specific expression of this receptor. A region that confers negative transcriptional regulation by cAMP maps to nucleotides -40 to -70 of this basal promoter. Using mobility shift and deoxyribonuclease footprinting assays, we also report the detection of Leydig cell-specific protein(s) that bind to the basal LH/CG receptor promoter. The binding of this protein(s) to the promoter involves an AP-2 consensus sequence beginning at nucleotide -59 as well as additional sequences that remain to be identified. In spite of the fact that the AP-2 site is involved, the protein-DNA complexes detected in Leydig cells are not recognized by an antibody to AP-2.