Reproduction cannot take place without the proper functioning of the lutropin/choriogonadotropin receptor (LHR). When the LHR does not work properly, ovulation does not occur in females and Leydig cells do not develop normally in the male. Also, because the LHR is essential for sustaining the elevated levels of progesterone needed to maintain pregnancy during the first trimester, disruptions in the functions of the LHR during pregnancy have catastrophic consequences. As such, a full understanding of the biology of the LHR is essential to the survival of our species. In this review we summarize our current knowledge of the structure, functions, and regulation of this important receptor.
Several clonal lines of cultured Leydig tumor cells have been established and characterized in terms of gonadotropin receptors and steroid production. Although freshly isolated cells derived from the M5480P tumor have functional hCG receptors, only two of the five clonal lines established were shown to bind significant quantities of hCG. In these clones, steroid production can be stimulated to the same extent by hCG, cholera toxin, and 8-Br-cAMP. The other three clones bind a small amount of hCG and respond to the hormone with a marginal increase in steroidogenesis. Steroid production, however, is significantly stimulated by cholera toxin or 8-Br-cAMP. A comparison of the steroids produced by freshly isolated cells and two of the clones revealed some changes in the steroidogenic pathway. The most obvious change is an increase in the ability of the cultured cells to synthesize 20 alpha-dihydroprogesterone (20 alpha-hydroxypregn-4-en-3-one). These clonal lines may provide a suitable model system for the study of gonadotropin actions and regulation of the expression of differentiated functions of Leydig cells.
A comparison of this review with the review on the LH/CG receptor that we published in this journal 4 yr ago (1) clearly shows that the field has advanced tremendously in this short period of time. Many of the questions that were unanswered then have now been conclusively answered. On the other hand, as is always the case, the new knowledge generated has also resulted in many new questions that are yet to be answered. Hopefully it is clear from this review that the knowledge and experimental tools generated during the last 4 yr have given us new ammunition to address such important issues as the elucidation of the structural determinants of the LH/CG receptor that are involved in the different functions of this receptor, as well as on the molecular bases of receptor activation, inactivation, and regulation. In fact, since we finished writing this review in September of 1992, we are certain that some of the questions left unanswered here will already have an answer in 1993 when this review is published.
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...
The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact. Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.
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