Prema Narayan

The luteinizing hormone receptor, LHCGR, is essential for fertility in males and females and genetic mutations in the receptor have been identified that result in developmental and reproductive defects. We have previously generated and characterized a mouse model (KiLHR^D582G) for familial male-limited precocious puberty caused by an activating mutation in the receptor. We demonstrated that the phenotype of the KiLHR^D582G male mice is an accurate phenocopy of male patients with activating LHCGR mutations. In this study, we observed that unlike women with activating LHCGR mutations who are normal, female KiLHR^D582G mice are infertile. Mice exhibit irregular estrous cyclicity, anovulation and precocious puberty. A temporal study from 2-24 wk of age indicated elevated levels of progesterone, androstenedione, testosterone and estradiol and upregulation of several steroidogenic enzyme genes. Ovaries of KiLHR^D582G mice exhibited significant pathology with the development of large hemorrhagic cysts as early as 3 wk of age, extensive stromal cell hyperplasia and hypertrophy with luteinization, numerous atretic follicles and granulosa cell tumors. Ovulation could not be rescued by the addition of exogenous gonadotropins. The body weights of the KiLHR^D582G mice were higher than wild type counterparts, but there was no increase in the body fat composition or metabolic abnormalities such as impaired glucose tolerance and insulin resistance. These studies demonstrate that activating LHCGR mutations do not produce the same phenotype in female mice as in humans and clearly illustrate species differences in the expression and regulation of LHCGR in the ovary, but not in the testis.

The LH receptor (LHR) is critical for steroidogenesis and gametogenesis. Its essential role is underscored by the developmental and reproductive abnormalities that occur due to genetic mutations identified in the human LHR. In males, activating mutations are associated with precocious puberty and Leydig cell hyperplasia. To generate a mouse model for the human disease, we have introduced an aspartic acid to glycine mutation in amino acid residue 582 (D582G) of the mouse LHR gene corresponding to the most common D578G mutation found in boys with familial male-limited precocious puberty (FMPP). In transfected cells, mouse D582G mLHR exhibited constitutive activity with a 23-fold increase in basal cAMP levels compared with the wild-type receptor. A temporal study of male mice from 7 days to 24 weeks indicated that the knock-in mice with the mutated receptor (KiLHR^D582G) exhibited precocious puberty with elevated testosterone levels as early as 7 days of age and through adulthood. Leydig cell-specific genes encoding LHR and several steroidogenic enzymes were up-regulated in KiLHR^D582G testis. Leydig cell hyperplasia was detected at all ages, whereas Sertoli and germ cell development appeared normal. A novel finding from our studies, not previously reported in the FMPP cases, is that extensive hyperplasia is commonly found around the periphery of the testis. We further demonstrate that the hyperplasia is due to premature proliferation and precocious differentiation of adult Leydig cells in the KiLHR^D582G testis. The KiLHR^D582G mice provide a mouse model for FMPP, and we suggest that it is a useful model for studying pathologies associated with altered LHR signaling.

A member of the G protein-coupled receptor superfamily, the LH receptor (LHR), and the two other glycoprotein hormone receptors are distinguished from the other members by the presence of a relatively large N-terminal extracellular domain that is responsible for high-affinity ligand binding. Transmembrane helix (TMH) 7 of LHR is amphipathic, with an extended face containing only hydrophobic side chains and another containing both hydrophobic and polar side chains with potential hydrogen bond donor and acceptor functions. Since several reports have shown the importance of this helix in ligand-mediated signaling, we have used Ala scanning mutagenesis to study eight amino acid residues of rat LHR that are invariant in the three glycoprotein hormone receptors, Leu⁵⁸⁶, Val⁵⁸⁷, Asn⁵⁹³, Ser⁵⁹⁴, Cys⁵⁹⁵, Asn⁵⁹⁷, Phe⁶⁰⁴, and Thr⁶⁰⁵. The wild type (WT) and mutant cDNAs were transiently transfected into COS-7 cells for characterization by human CG (hCG) binding and cAMP production. No differences were detected in dissociation constants (K_ds) or basal cAMP production relative to WT LHR, but three categories of LHR mutants were distinguished from WT LHR based upon their expression levels and responsiveness to hCG: 1) comparable or higher expression but reduced ligand responsiveness (N593A and C595A), 2) reduced expression and ligand responsiveness (N597A and T605A), and 3) comparable expression and responsiveness (L586A, V587A, S594A, and F604A). Three other mutants, C595M, F604Y, and T605Y, were comparable to WT LHR in ligand responsiveness. To provide more information on Asn⁵⁹³ and Asn⁵⁹⁷, a total of 12 replacements were investigated. Of considerable interest and potential significance was the finding that many of the replacements in LHR resulted in either loss of function (N593A, Q, S; N597R) or gain of function (N593R and N597Q), this being the first evidence of a position in LHR that, depending upon the nature of the amino acid residue, can result in constitutive activation and/or diminished responsiveness to ligand. The results of molecular modeling and energy minimization of TMHs 6 and 7, based on a postulated interaction between Asp⁵⁵⁶ (TMH 6) and Asn⁵⁹³/Asn⁵⁹⁷ (TMH 7), indicated that, while there is not a correlation between function and predicted energies of WT LHR and the mutants, reorientation of one or both helices is responsible for the functional changes observed. Possible interactions of TMHs 3 and 4 and of 5 and 6 were suggested by molecular modeling. Ten mutants were prepared of two amino acid residues that are invariant in the glycoprotein hormone receptors and have side chain hydrogen bond donor and acceptor function, Glu⁴²⁹ in TMH 3 and Asn⁵¹³ in TMH 5. Expression levels and hCG-mediated signaling were reduced in most of the LHR mutants, but none of these exhibited constitutive receptor activation. We conclude that Glu⁴²⁹ is not critical for receptor function, while Asn⁵¹³ appears to be particularly important in receptor folding and/or trafficking. The results reported herein indicate an important role for TMH 7, and particularly Asn⁵⁹³ and Asn⁵⁹⁷, in the process of receptor activation. Moreover, these two asparagines, although in close proximity to each other in TMH 7, are quite distinct in function as evidenced by certain replacements that can lead to loss of function in one and gain of function in the other.