Expression and localization of luteinizing hormone receptor in the female mouse reproductive tract

The presence of the LH receptor (LHR) in nongonadal tissues of the reproductive tract has been reported, but localization studies have not been performed. Our objectives were to demonstrate the presence of LHR in the reproductive tract and to localize receptor expression. Reproductive age rats and mice were obtained and (125)I-hCG binding assays were performed on membrane preparations from the uterus, ovary, liver, and testis. In situ hybridizations were performed using (35)S-labeled antisense and sense RNA probes prepared from nucleotides 1-591 of the mouse LHR cDNA. Specific hCG binding was detected in membrane preparations from the ovary, uterus, and testis but not in the liver in both the rat and mouse. In the ovary, LHR mRNA was localized in theca cells, large follicles, and corpora lutea as expected. In the uterus, LHR mRNA was expressed in stromal cells of the endometrium and in the uterine serosa. Uterine smooth muscle cells had low levels of expression, and the endometrial epithelium was negative. In the oviduct, high levels of LHR expression were noted on the serosa and in subepithelial cells. Oviductal smooth muscle had low expression, and the epithelium was negative. We conclude that functional, nongonadal LHR are expressed in the mouse reproductive tract. The presence and localization of LHR expression in the mouse reproductive tract lay the foundation for transgenic models to address the physiologic role of these receptors.     

The extracellular domain of luteinizing hormone receptor dictates its efficiency of maturation

The processing of luteinizing hormone receptor (LHR) shows marked differences in different species. While the human LHR is predominantly expressed as the mature, 90 kDa species, rat LHR exists mostly in the 70 kDa precursor form. Since the extracellular domain of the LHR is unusually large in comparison with other G protein-coupled receptors, the present studies examined the role of extracellular domain in its processing. FLAG-tagged chimeric LH receptors were constructed by substituting the extracellular domain of the human receptor in rat LHR (hrr) and the extracellular domain of the rat receptor in human LHR (rhh). The intracellular processing, ligand binding and recycling of the chimeric receptors were compared with that of the wild type receptors in 293T cells. The results showed that the human and rat LHR were expressed predominantly as 90 and 70 kDa species, respectively, as expected. The introduction of the rat extracellular domain into the human LHR (rhh) decreased the abundance of the mature form with an increase in the precursor form. Conversely, substitution of the extracellular domain of the rat LHR by the extracellular domain of the human LHR (hrr) led to an increase in the mature form with a corresponding decrease in the precursor form. Changes were also observed in the ligand binding and recycling of the wild type and chimeric receptors. These results suggest that the extracellular domain of the LHR is one of the determinants that confer its ability for proper maturation and cell surface expression.     

Identification and structural characterization of the neuronal luteinizing hormone receptor associated with sensory systems

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor involved in regulation of ovarian and testicular functions. Here we show that the receptor is present also in specific areas of the peripheral and central nervous system and may thus have a broader functional role than has been anticipated. Full-length LHR mRNA and two receptor protein species of M(r) 90,000 and 73,000, representing mature and precursor forms, respectively, were expressed in adult and developing rat nervous tissue, starting at fetal day 14.5. The receptor was capable of ligand binding because it was purified by ligand affinity chromatography, and human chorionic gonadotropin and LH were able to displace (125)I-labeled human chorionic gonadotropin binding to fetal head membranes in a dose-dependent manner. Finally, two 5'-flanking sequences ( approximately 2 and 4 kb) of the rat LHR gene were shown to direct expression of the lacZ reporter to specific areas of the peripheral and central nervous system in fetal and adult transgenic mice, especially to structures associated with sensory, memory, reproductive behavior, and autonomic functions. Importantly, the transgene activity was confined to neurons and colocalized with the cytochrome P450 side chain cleavage enzyme. Taken together, these results indicate that the neuronal LHR is a functional protein, implicating a role in neuronal development and function, possibly by means of regulating synthesis of neurosteroids.     

Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor.

To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage, we sought to convert the noncleaving luteinizing hormone (LH) receptor (LHR) into a cleaved, two-subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125I-labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N-linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which has only one N-linked glycan. Elimination by mutagenesis of the most strategic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavage. Removal of the other N-linked glycans had no additive effect. A much greater degree of cleavage ( approximately 50%) was evident in a chimeric LH-TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317-367) was replaced with the corresponding domain of the TSHR (residues 363-418). Similarly cleaving LHR were created using a much smaller component within this region, namely LHR-NET317-319 replaced with TSHR-GQE367-369, or by substitution of the same three amino-acid residues with AAA (LHR-NET317-319AAA). In summary, our data alter current concepts regarding TSHR cleavage by suggesting limited (not absent) amino-acid specificity in a region important for TSHR cleavage (GQE367-369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.     

Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells.

The murine receptor for luteinizing hormone (LHR) was cloned and expressed in L cells. This LHR (mature protein of 674 amino acids) is very similar to that of the rat (same length, 36 amino acid differences) but differs significantly more from that of man (673 amino acids, 109 differences). Expression of the murine LHR in L cells led to the appearance of binding sites for human chorionic gonadotropin (hCG) with a Kd of 150 pM and an LH- and hCG-stimulable adenylyl cyclase activity (EC50 = 50-100 pM hCG). Upon labeling pools of phosphoinositides with [3H]myo-inositol, L cells expressing the murine LHR responded to hCG with an increase in their rate of phosphoinositide hydrolysis (EC50 = 2,400 pM hCG). This was accompanied by an increase in intracellular Ca2+ [( Ca2+]i), as determined by the Fura2 method. This increase in [Ca2+]i in response to hCG was dependent on the LHR, for HCG did not affect [Ca2+]i in L cells not expressing the LHR. The effect was not due to the cAMP-forming activity of the LH receptor, for neither forskolin nor prostaglandin E1, which both increase cAMP levels in L cells, had a similar effect in either control or LHR-expressing cells and isoproterenol had no effect in L cells expressing a functionally active hamster beta-adrenergic receptor. The effect was also not due to overexpression of a Gs-coupled receptor, for L cells expressing 8-fold higher levels of the human V2 vasopressin receptor did not mimic the Ca(2+)-mobilizing response of the LH receptor. We conclude that the LH receptor has the capability of activating two intracellular signaling pathways: one leading to stimulation of adenylyl cyclase and resulting in increases in cAMP and a second leading to stimulation of phospholipase C and resulting in formation of inositol phosphates and elevations in [Ca2+]i. These data correlate positively with and provide a mechanistic explanation for previous reports on the ability of hCG to mobilize phosphoinositides and increasing [Ca2+]i in luteal and granulosa cells (e.g. Davis, J. S., West, L. A., and Farese, R. V. (1984) J. Biol. Chem. 259, 15028-15034).     

Expression and regulation of mRNAs for insulin-like growth factor-I receptor and LH receptor in corpora lutea

Relationship between insulin-like growth factor-I receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF2 inhibited expression of IGF-IR and LHR. PGE2 negated the inhibiting effects of PGF2. These data suggest that IGF-I may be involved in regulating CL function, and maintaining CL structure through changes in expression of its receptors. Inhibited expression of IGF-IR by PGF2 may be part of mechanisms for regression of CL.     

Evidence that palmitoylation of carboxyl terminus cysteine residues of the human luteinizing hormone receptor regulates postendocytic processing

Palmitoylation is a well-conserved posttranslational modification among members of the G protein-coupled receptor superfamily. The present study examined the role of palmitoylation in endocytosis and postendocytic trafficking of the human LH receptor (LHR). Palmitoylation of the LHR was determined by incorporation of [3H]palmitic acid into wild-type (WT) or mutant receptor in which the potential palmitoylation sites, C643 and C644, were mutated to glycine residues. The WT receptor showed incorporation of [3H]palmitic acid into the mature 90-kDa form of the receptor whereas mutation of the two Cys residues abrogated this incorporation, indicating that Cys 643 and C644 are the sites of palmitoylation. The role of palmitoylation on endocytosis and postendocytic processing was examined by testing the ability of the WT and mutant receptor to undergo internalization, recycling, and lysosomal degradation. Compared with the WT receptor, the mutant receptor showed increased internalization and decreased recycling, suggesting that retention of palmitic acid residues at Cys 643 and 644 promotes LHR recycling. The role of palmitoylation on receptor recycling was substantiated by demonstrating that a different mutant, D578H LHR, which is deficient in palmitoylation, also recycled less efficiently. Furthermore, the data show that palmitoylation, not the rate of internalization, determines the efficiency of recycling. The present study shows that palmitoylation of cysteine residues 643 and 644 of the human LHR is a determinant of recycling.