Impact of subdomain D1 of the short form S1b of the human prolactin receptor on its inhibitory action on the function of the long form of the receptor induced by prolactin.

BACKGROUND: Long-form (LF) homodimers of the human prolactin receptor (PRLR) mediate prolactin's diverse actions. Short form S1b inhibits the LF function through heterodimerization. Reduced S1b/LF-ratio in breast cancer could contribute to tumor development/progression. Current work defines the structural and functional relevance of the D1 domain of S1b on its inhibitory function on prolactin-induced LF function. METHODS: Studies were conducted using mutagenesis, promoter/signaling analyses, bioluminescence resonance energy transfer (BRET) and molecular modeling approaches. RESULTS: Mutation of E69 in D1 S1b or adjacent residues at the receptor surface near to the binding pocket (S) causes loss of its inhibitory effect while mutations away from this region (A) or in the D2 domain display inhibitory action as the wild-type. All S1b mutants preserved prolactin-induced Jak2 activation. BRET reveals an increased affinity in D1 mutated S1b (S) homodimers in transfected cells stably expressing LF. In contrast, affinity in S1b homodimers with either D1 (A) or D2 mutations remained unchanged. This favors LF mediated signaling induced by prolactin. Molecular dynamics simulations show that mutations (S) elicit major conformational changes that propagate downward to the D1/D2 interface and change their relative orientation in the dimers. CONCLUSIONS: These findings demonstrate the essential role of D1 on the S1b structure and its inhibitory action on prolactin-induced LF-mediated function. GENERAL SIGNIFICANCE: Major changes in receptor conformation and dimerization affinity are triggered by single mutations in critical regions of D1. Our structure-function/simulation studies provide a basis for modeling and design of small molecules to enhance inhibition of LF activation for potential use in breast cancer treatment.

Intramolecular disulfide bonds of the prolactin receptor short form are required for its inhibitory action on the function of the long form of the receptor.

The short form (S1b) of the prolactin receptor (PRLR) silences prolactin-induced activation of gene transcription by the PRLR long form (LF). The functional and structural contributions of two intramolecular disulfide (S-S) bonds within the extracellular subdomain 1 (D1) of S1b to its inhibitory function on the LF were investigated. Mutagenesis of the paired cysteines eliminated the inhibitory action of S1b. The expression of the mutated S1b (S1bx) on the cell surface was not affected, indicating native-like folding of the receptor. The constitutive JAK2 phosphorylation observed in S1b was not present in cells expressing S1bx, and JAK2 association was disrupted. BRET(50) (BRET(50) represents the relative affinity as acceptor/donor ratio required to reach half-maximal BRET [bioluminescence resonance energy transfer] values) showed decreased LF/S1bx heterodimeric-association and increased affinity in S1bx homodimerization, thus favoring LF homodimerization and prolactin-induced signaling. Computer modeling based on the PRLR crystal structure showed that minor changes in the tertiary structure of D1 upon S-S bond disruption propagated to the quaternary structure of the homodimer, affecting the dimerization interface. These changes explain the higher homodimerization affinity of S1bx and provide a structural basis for its lack of inhibitory function. The PRLR conformation as stabilized by S-S bonds is required for the inhibitory action of S1b on prolactin-induced LF-mediated function and JAK2 association.

EAR2 and EAR3/COUP-TFI regulate transcription of the rat LH receptor.

Our previous studies demonstrated regulation of the human LH receptor (hLHR) promoter by nuclear orphan receptors EAR2, EAR3/COUP-TFI (repression), and TR4 (activation) through a direct-repeat motif (hDR). The current studies investigated the differential binding of orphan receptors to rat (rLHR) and hLHR promoters, and their modulation of rLHR gene transcription in rat granulosa cells. The rLHR DR with one nucleotide difference from hDR at its core sequence mediated inhibition of the rLHR transcription, to which EAR2 and EAR3/COUP-TFI but not TR4 bound. The A/C mismatch was responsible for the lack of TR4 binding and function, but had no effect on EAR2 and EAR3/COUP-TFI. EAR2 and EAR3/COUP-TF bound to the rLHR DR with lower affinity than to the hDR, and exhibited lesser inhibitory capacity. This difference resulted from the lack of a guanine in the rDR, which is present 3' next to the hDR core. These studies have identified sequence-specific requirements for the binding of EAR2, EAR3/COUP-TFI, and TR4 to the DRs that explain their differential regulation of rat and human LHR genes. In addition, hCG treatment significantly reduced the inhibition of rLHR gene in granulosa cells and also decreased EAR2 and EAR3/COUP-TFI protein levels. These results indicate that hormonally regulated expression of EAR2 and EAR3/COUP-TFI contributes to gonadotropin-induced derepression of LHR promoter activity in granulosa cells.

Isolation and characterization of two novel forms of the human prolactin receptor generated by alternative splicing of a newly identified exon 11.

We have identified a novel exon 11 of the human prolactin receptor (hPRLR) gene that is distinct from its rodent counterparts and have demonstrated the presence of two novel short forms of the hPRLR (S1(a) and S1(b)), which are derived from alternative splicing of exons 10 and 11. S1(a) encodes 376 amino acids (aa) that contain partial exon 10 and a unique 39-aa C-terminal region encoded by exon 11. S1(b) encodes 288 aa that lack the entire exon 10 and contains 3 amino acids at the C terminus derived from exon 11 using a shifted reading frame. These short forms, which were found in several normal tissues and in breast cancer cell lines, were expressed as cell surface receptors and possessed binding affinities comparable with the long form. Unlike the long form, neither short form was able to mediate the activation of the beta-casein gene promoter induced by prolactin. Instead they acted as dominant negative forms when co-expressed with the long form in transfected cells. Due to a marked difference in the cellular levels between the two short forms in transfected cells, S1(b) was more effective in inhibiting the prolactin-induced activation of the beta-casein gene promoter mediated by the long form of the receptor. The low cellular level of S1(a) was due to its more rapid turnover than the S1(b) protein. This is attributable to specific residues within the C-terminal unique 39 amino acids of the S1(a) form and may represent a new mechanism by which the hPRLR is modulated at the post-translational level. Since both short forms contain abbreviated cytoplasmic domains with unique C termini, they may also exhibit distinct signaling pathways in addition to modulating the signaling from the long form of the receptor. These receptors may therefore play important roles in the diversified actions of prolactin in human tissues.

Testicular endocrine function in GH receptor gene disrupted mice.

The consequences of disruption of GH receptor gene in GH receptor knockout mice on testicular function were evaluated. Adult male GH receptor knockout mice and their normal siblings were divided in to two subgroups and treated with either saline or ovine LH (0.3 microg/g BW) in saline. One hour after saline or LH administration, blood was obtained via heart puncture. Plasma IGF-I, LH, FSH, PRL, androstenedione, and testosterone levels were measured by RIAs. Testicular LH and PRL receptor numbers as well as pituitary LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were measured. Also, testicular morphometric analysis was performed. Unlike in normal, wild-type mice, the circulating IGF-I was undetectable in GH receptor knockout mice. The plasma PRL levels were (P<0.01) higher in GH receptor knockout mice than in their normal siblings. The basal LH secretion was similar in normal and GH receptor knockout mice. However, the circulating FSH levels were lower (P<0.001) in GH receptor gene disrupted mice. Administration of LH resulted in a significant (P<0.001) increase in plasma testosterone levels in both GH receptor knockout and normal mice. However, this testosterone response was attenuated (P < 0.01) in GH receptor knockout mice. Plasma androstenedione responses were similar in both GH receptor knockout and normal mice. Testicular LH and PRL receptor numbers were significantly decreased in GH receptor knockout mice. The results of the morphometric analysis of the testis revealed that the Leydig cell volume per testis was reduced in mice with GH receptor gene disruption. The steady-state of LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were not different in GH receptor knockout mice relative to their normal siblings. The present in vivo study demonstrates that in GH receptor knockout mice, LH action on the testis in terms of testosterone secretion is significantly attenuated and suggests that this is due to a decrease in the number of testicular LH receptors. The reduced number of PRL receptors may contribute to the diminished responsiveness of testicular steroidogenesis to LH by decreased ability to convert androstenedione to testosterone. These changes are most likely due to the absence of circulating IGF-I. These findings provide evidence that systemic IGF-I plays a major modulatory role in testicular endocrine function.

Regulation of steroidogenic enzymes and a novel testicular RNA helicase.

Luteinizing hormone (LH) supports steroidogenesis and maintains testicular and ovarian function. Mediators of LH action exert homologous regulation of membrane receptors, steroidogenic enzymes and other regulatable genes of the Leydig cell (LC). Androgen and estrogen induced by LH could act through its cognate receptors in the LC to regulate gene expression. Although androgens are unquestionable essential for spermatogenesis and presumably exert their heterologous action through androgen receptors present in the Sertoli its regulatory mechanism in germinal cell maturation is far from clear. In contrast to physiological concentrations of gonadotropins which maintain the steroidogenic functions and LH and prolactin receptors in the gonads, high concentrations of gonadotropin (hCG) cause receptor down-regulation and desensitization of steroidogenic enzymes of the LCs in vivo (3beta-hydroxysteroid dehydrogenase types I and II, 17alpha-hydroxylase/17,20 lyase, and 17beta-hydroxysteroid dehydrogenase type III [17beta-HSD]). In addition, 17beta-HSD is regulated by compartmentalized endogenous glucose/ATP. The attenuation of steroidogenesis which results from receptor mediated activation by cognate hormone, but is independent of the subsequent phase of receptor down-regulation, is due to changes at the transcriptional level. Among the candidates affecting this regulation are active steroid metabolites (direct or indirect of steroids and other mediator(s) i.e. cAMP, putative transcription factors induced by LH action). Differential display assay revealed another gene which is transcriptionally regulated by gonadotropin termed GRTH (Gonadotropin Regulated Testicular Helicase). GRTH is a novel member of the DEAD-box family of RNA helicases, and is specifically expressed in LCs and meiotic LC of the testis. It is markedly up-regulated by hCG via cAMP-induced androgen formation in LCs at doses that cause down-regulation of receptors and steroidogenic enzymes. GRTH functions as a translational activator. Androgen produced by gonadotropin stimulation exerts intracrine/autocrine actions on GRTH, and also could influence transcription within the seminiferous tubule. GRTH may contribute to the control of steroidogenesis, including the restoration of down regulated cellular functions, and in the paracrine regulation of androgen dependent gene(s) involved in the meiotic process, and could thus have a crucial role in spermatogenesis.

Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase.

A previously unidentified gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) was cloned and characterized as a 79-kDa cytoplasmic protein expressed in Leydig cells of the rat testis. GR-LACS shares sequence identity with two conserved regions of the LACS and luciferase families, including the ATP/AMP binding domain and the 25-aa fatty acyl-CoA synthetase signature motif, but displays low overall amino acid similarities (23-28%). GR-LACS mRNA is expressed abundantly in Leydig cells of the adult testis and to a lesser degree in the seminiferous tubules in spermatogonia and Sertoli cells. It is also observed in ovary and brain. Immunoreactive protein expression was observed mainly in Leydig cells and minimally in the tubules but was not detected in other tissues. In vivo, treatment with a desensitizing dose of human chorionic gonadotropin caused transcriptional down-regulation of GR-LACS expression in Leydig cells. The expressed protein present in the cytoplasm of transfected cells displayed acyl-CoA synthetase activity for long chain fatty acid substrates. GR-LACS may contribute to the provision of energy requirements and to the biosynthesis of steroid precursors and could participate through acyl-CoA's multiple functions in the regulation of the male gonad.

Nuclear orphan receptors regulate transcription of the gene for the human luteinizing hormone receptor.

An imperfect estrogen receptor half-site response element direct-repeat, located within the TATA-less promoter of the human luteinizing hormone receptor (hLHR), was identified as an inhibitory site for Sp1/Sp3-driven basal transcription. Isolation of proteins recognizing this site by yeast one-hybrid screening of a human placenta cDNA library revealed three nuclear orphan receptors, EAR2, EAR3/COUP-TFI, and TR4. Electrophoresis mobility shift assays demonstrated that the in vitro translated nuclear orphan receptors specifically bound the direct-repeat motif of the hLHR promoter. Also, endogenous EAR2 and EAR3/COUP-TFI from JAR cell and human testis and TR4 from testes bound this motif in electrophoresis mobility shift assays. Functional analyses in CV-1 cells showed that EAR2 and EAR3/COUP-TFI repressed the hLHR promoter activity by up to 70% in a dose-dependent and sequence-specific manner. Conversely, TR4 activated the hLHR promoter activity up to 2.5-fold through binding to the same cis-element. The stimulation was reversed by coexpression of EAR2 or EAR3/COUP-TFI, indicating their competitive binding for this site. Such recognition of a common cognate site by the proteins with antagonistic functions implies that a net regulation of the hLHR gene may result from the relative availability of repressors and activator in a physiological state. This also may contribute to the differential expression of the hLHR gene in gonadal and non-gonadal tissues.

A novel gonadotropin-regulated testicular RNA helicase. A new member of the dead-box family.

A gonadotropin-regulated testicular RNA helicase (GRTH) was identified and characterized. GRTH cloned from rat Leydig cell, mouse testis, and human testis cDNA libraries is a novel member of the DEAD-box protein family. GRTH is transcriptionally up-regulated by chorionic gonadotropin via cyclic AMP-induced androgen formation in the Leydig cell. It has ATPase and RNA helicase activities and increases translation in vitro. This helicase is highly expressed in rat, mouse, and human testes and weakly expressed in the pituitary and hypothalamus. GRTH is produced in both somatic (Leydig cells) and germinal (meiotic spermatocytes and round haploid spermatids) cells and is developmentally regulated. GRTH predominantly localized in the cytoplasm may function as a translational activator. This novel helicase could be relevant to the control of steroidogenesis and the paracrine regulation of androgen-dependent spermatogenesis in the testis.

Expression of functional leptin receptors in rodent Leydig cells.

Several studies indicate that the size of body fat stores and the circulating levels of the adipocyte-derived hormone leptin are able to influence the activity of the hypothalamic-pituitary-gonadal axis. The leptin-hypothalamic-pituitary-gonadal interactions have been mainly studied at the level of the central nervous system. In this study, we investigated the possibility that leptin may have direct effects on the rodent Leydig cell function. To probe this hypothesis, we first analyzed the expression of leptin receptors (OB-R) in rodent Leydig cells in culture. RT-PCR studies showed that rat Leydig cells express both the long (OB-Rb) and short isoform (OB-Ra) of leptin receptor, whereas MLTC-1 cells (a murine Leydig tumor cell line) express only the long isoform. Short-term (30-90 min) incubation of rat Leydig cells with increasing concentrations ofleptin (2-500 ng/ml) led to a significant and dose-dependent inhibition of human (h)CG-stimulated testosterone (T) production (approximately 60% reduction, IC50 = 20 ng/ml) but no change in basal androgen release. Also, leptin (150 ng/ml) amplified hCG-induced intracellular cAMP formation (1- to 2-fold) without modifying basal cAMP levels. Subsequent experiments showed that leptin inhibited 8Br-cAMP-stimulated T production, indicating that leptin's effect is exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T secretion was accompanied by a significant reduction of androstenedione and a concomitant rise of the precursor metabolites pregnenolone, progesterone, and 17-OH-progesterone, conceivable with a leptin-induced lesion of 17,20 lyase activity. Separate experiments performed with the MLTC-1 cells (not expressing cytochrome P450-17alpha) showed that leptin, though amplifying hCG-stimulated cAMP production, did not modify hCG-stimulated pregnenolone and progesterone release. These results further indicate that leptin action on steroidogenesis occurs downstream of progesterone synthesis. Northern Blot experiments showed no acute effect of leptin on cytochrome P450-17alpha messenger RNA accumulation in rat Leydig cells in basal and hCG-stimulated conditions, excluding that the rapid changes observed were caused by messenger RNA degradation. In conclusion, these findings, for the first time, show that leptin has direct, receptor-mediated actions on rodent Leydig cells in culture, at concentrations within the range of obese men.

The human luteinizing hormone receptor gene promoter: activation by Sp1 and Sp3 and inhibitory regulation.

To understand the transcriptional mechanism(s) of human LH receptor (LHR) gene expression, we have identified the dominant functional cis-elements that regulate the activity of the promoter domain (-1 to -176 bp from ATG). Mutagenesis demonstrated that the promoter activity was dependent on two Sp1 domains (-79 bp, -120 bp) in a transformed normal placental cell (PLC) and the choriocarcinoma JAR cell. Both elements interacted with endogenous Sp1 and Sp3 factors but not with Sp2 or Sp4. In Drosophila SL2 cells, the promoter was activated by either Sp1 or Sp3. An ERE half-site (EREhs) at -174 bp was inhibitory (by 100%), but was unresponsive to estradiol and did not bind the estrogen receptor or orphan receptors ERR1 and SF-1. The 5' upstream sequence (-177 to -2056 bp) inhibited promoter activity in PLC by 60%, but only minimally in JAR cells. Activation of the human LHR promoter through Sp1/3 factors is negatively regulated through EREhs and upstream sequences to exert control of gene expression.

The rat 17beta-hydroxysteroid dehydrogenase type III: molecular cloning and gonadotropin regulation.

17Beta-hydroxysteroid dehydrogenase (17betaHSD), the enzyme that catalyzes the final step of testosterone biosynthesis in the testis, was cloned from a rat Leydig cell complementary DNA library to gain insights into the functional requirements, activation mechanisms, and molecular regulation. The 17betaHSD complementary DNA encoded 306 amino acids (molecular mass of 33.7 kDa) and displayed 75% and 85% amino acid sequence homology to the human and mouse 17betaHSD type III enzymes, respectively. Northern analysis revealed a single 1.4-kb messenger RNA (mRNA) species in rat Leydig cells, whereas ovarian mRNA was detected only by RT-PCR amplification. The cloned 17betaHSD expressed in mammalian cell lines specifically catalyzed the reductive reaction in androgen formation with androstenedione as the preferred substrate. This reaction was significantly reduced in the absence of glucose. Expression of the endogenous 17betaHSD gene in rat Leydig cells was inhibited by a single dose of hCG in vivo, with maximum reduction of steady state mRNA levels at 24 h and recovery at 9 days. Such agonist-induced down-regulation of 17betaHSD expression, which preceded the marked reduction of LH receptors, resulted from changes at the transcriptional level and was accompanied by loss of enzymatic activity. These studies have demonstrated a glucose requirement for optimal activity of the enzyme in vitro and for a role of gonadotropin in regulating the expression of 17betaHSD gene in vivo. Cloning of the 17betaHSD type III enzyme from rat Leydig cells will facilitate further investigation of the molecular regulation of its activity in the testis.

The human prolactin receptor gene structure and alternative promoter utilization: the generic promoter hPIII and a novel human promoter hP(N).

The 5'-untranslated region of the human prolactin receptor (hPRLR) gene contains two alternative first exons, hE1(3), the human counterpart of the rat and mouse E1(3) and a novel human type of alternative first exon termed hE1N, also a common non-coding exon 2 and a third exon containing the translation initiation codon. hE1(3) was localized approximately 800 bp 5' from the hE1N in the genome. The two distinct first exons hE1(3) and hE1N are expressed in human breast tissue, breast cancer cells, gonads and liver. Overall, the transcript containing hE1(3) is prevalent in most tissues. The coding region of the gene comprises eight exons (exon 3-10), in which exon 10 encodes most of the intracellular domain. hE1(3) and hE1N are transcribed from alternative promoters hPIII and hP(N), respectively. The hPIII, containing identical Sp1 and C/EBP elements as in the rodent promoters, shares 81% similarity in the region -480/-106 to both the rat and mouse. The novel promoter hP(N) contains putative binding sites for ETS-family proteins and a half-site for nuclear receptors. Therefore, both promoters likely utilize distinct mechanisms in controlling the hPRLR gene transcription. The different promoter utilization of the hPRLR gene in diverse tissues may confer differential prolactin response through activation of different promoters.

Genomic distribution and gonadal mRNA expression of two human luteinizing hormone receptor exon 1 sequences in random populations.

Exon 1 of the human luteinizing hormone receptor (LHR) gene coding region exhibits at least two forms of sequence heterogeneity between 37 and 60 bp, spanning the junction of the signal peptide and the amino terminus of the mature protein. The LHR 1 differs from the LHR 2 by the insertion of 6 bp in exon 1 but is of identical sequence in the 5' flanking region. RFLP analysis of the two haplotypes within a random population of 63 individuals revealed allele frequencies of 0. 37 and 0.63 for LHR 1 and LHR 2, respectively. 94% of the samples contained at least one LHR 2 allele, whereas only 68% contained the LHR 1 allele. No gender differences were observed, and both homozygotes and heterozygotes displayed apparently normal reproduction. Reverse-transcriptase polymerase chain-reaction analyses of LHR mRNA from testes and ovaries revealed that both haplotypes are transcribed in normal individuals, with no difference in tissue specific distribution. Thus, at least two functional polymorphic forms of exon 1 coding region of the same LHR gene are present in a random human population.

Regulation of 3beta-hydroxysteroid dehydrogenase in gonadotropin-induced steroidogenic desensitization of Leydig cells.

3Beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerases (3beta-HSD) are enzymes that catalyze the conversion of delta5 to delta4 steroids in the gonads and adrenal for the biosynthesis of sex steroid and corticoids. In gonadotropin-desensitized Leydig cells, from rats treated with high doses of human CG (hCG), testosterone production is markedly reduced, a finding that was attributed in part to reduction of CYP17 expression. In this study, we present evidence for an additional steroidogenic lesion induced by gonadotropin. Using differential display analysis of messenger RNA (mRNA) from Leydig cells of rats treated with a single desensitizing dose of hCG (2.5 microg), we found that transcripts for type I and type II 3beta-HSD were substantially (5- to 8-fold) down-regulated. This major reduction, confirmed by RNase protection assay, was observed at the high hCG dose (2.5 microg), whereas minor or no change was found at lower doses (0.01 and 0.1 microg). In contrast, 3beta-HSD mRNA transcripts were not changed in luteinized ovaries of pseudopregnant rats treated with 2.5 microg hCG. The down-regulation of 3beta-HSD mRNA in the Leydig cell resulted from changes at the transcriptional level. Western blot analysis showed 3beta-HSD protein was significantly reduced by hCG treatment, with changes that were coincidental with the reduction of enzyme activity and temporally consistent with the reduction of 3beta-HSD mRNA but independent of LH receptor down-regulation. The reduction of 3beta-HSD mRNA resulting from transcriptional inhibition of gene expression, and the consequent reduction of 3beta-HSD activity could contribute to the inhibition of androgen production in gonadotropin-induced steroidogenic desensitization of Leydig cells. The gender-specific regulation of 3beta-HSD by hCG reflects differential transcriptional regulation of the enzymes to accommodate physiological hormonal requirements and reproductive function.

Transcriptional regulation of the generic promoter III of the rat prolactin receptor gene by C/EBPbeta and Sp1.

Three promoters are operative in the rat prolactin receptor gene as follows: promoter I (PI) and II (PII) are specific for the gonads and liver, respectively, and promoter III (PIII) is common to several tissues. To investigate the mechanisms controlling the activity of promoter III, its regulatory elements and transcription factors were characterized in gonadal and non-gonadal cells. The TATA-less PIII domain was localized to the region -437 to -179 (ATG +1) containing the 5'-flanking region and part of the non-coding first exon. Within the promoter domain, a functional CAAT-box/enhancer binding protein (C/EBP) (-398) and an Sp1 element (-386), which bind C/EBPbeta and Sp1/Sp3, respectively, contribute individually to promoter activation in gonadal and non-gonadal cells. However, significant redundancy was demonstrated between these elements in non-gonadal cells. Additionally, an element within the non-coding exon 1 (-338) is also required for promoter activity. Activation of PIII by the widely expressed Sp1 and C/EBPbeta factors explains its common utilization in multiple tissues. Moreover, whereas the rat and mouse PIII share similar structure and function, the mouse PI lacks the functional SF-1 element and hence is inactive. These findings indicate that promoter III is of central importance in prolactin receptor gene transcription across species.

The luteinizing hormone receptor.

The luteinizing hormone receptor (LHR) is a member of the subfamily of glycoprotein hormone receptors within the superfamily of G protein-coupled receptor (GPCR)/seven-transmembrane domain receptors. Over the past eight years, major advances have been made in determining the structure and function of the LHR and its gene. The hormone-binding domain has been localized to exons 1-7 in the extracellular (EC) domain/region of the receptor, which contains several leucine-rich repeats. High-affinity binding of LH and human chorionic gonadotrophin (hCG) causes secondary hormone or receptor contacts to be established with regions of the EC loop/transmembrane module that initiate signal transduction. Models of hormone-receptor interaction have been derived from the crystal structures of hCG and of the ribonuclease inhibitor, which also contains leucine-rich repeats. Such models provide a framework for the interpretation of mutational studies and for further experiments. The extracellular domain of the receptor has been overexpressed in vitro, which will facilitate crystallographic resolution of the structure of the receptor-binding site. The transmembrane domain/loop/cytoplasmic module transduces the signal for coupling to G proteins. Several constitutive, activating mutations that cause human disease have been found in helix VI and adjacent structures. These mutations have provided valuable information about mechanisms of signal transfer and G protein coupling. The structure of the LHR gene has been elucidated, and the regulation of its transcription is beginning to be understood. Valuable insights into receptor evolution have been derived from analysis of sequence homologies, the gene structure of glycoprotein hormone receptors and other members of the GPCR family, and the glycoprotein hormone receptor-like precursors identified in several invertebrate species.

A novel human luteinizing hormone receptor gene.

A novel human luteinizing hormone receptor (LHR) gene was isolated from a human placental genomic library. This gene (Gene II) differs from that previously isolated from a lymphocyte library (Gene I) by several base changes in the 5' flanking region and the deletion of 6 nucleotides in the coding region (+55 to +60). The sequence of the exon 1 coding region of gene II conforms to the sequence of the human ovarian LHR cDNA. Furthermore, primer extension and reporter gene analyses indicate that both the Gene II promoter and the human ovarian LHR mRNA transcriptional start sites are located within the -176 bp TATA-less 5' flanking domain. Additional upstream transcriptional start sites (> -176 bp) were identified in human testicular mRNA and the human choriocarcinoma JAR cell. Restriction enzyme analysis verifies that both LHR genes are present in the human genome, and gene dosing reveals four copies of the human LHR in contrast to a single copy in the rat genome. Chromosomal mapping localizes all copies of the human LHR to the chromosome 2p16-21 loci. These studies suggest that tissue-specific LHR promoter utilization and LHR gene expression may be correlated with gene diversity.

Prolactin receptor gene diversity: structure and regulation.

The diverse functionality of prolactin and the wide expression of the prolactin receptor suggest a complex system regulated by this polypeptide hormone. Different hormone and receptor forms, as well as differential signal transduction pathways, contribute to the functional diversity of prolactin's actions. The heterogeneity of rat prolactin receptor gene transcripts in their 5'-untranslated region has led to the recognition of multiple and tissue-specific utilization of prolactin receptor gene promoters in gonadal and non-gonadal tissues. These findings have provided insights into the molecular bases for the diversity of prolactin's actions. It is now clear that cellular responsiveness to prolactin can be regulated through differential promoter control of the expression of the surface receptors for prolactin in different target tissues.

Steroidogenic factor-1 is an essential transcriptional activator for gonad-specific expression of promoter I of the rat prolactin receptor gene.

The expression of the prolactin receptor is under the control of two putative tissue-specific (PI, gonads; PII, liver) and one common (PIII) promoters (Hu, Z. Z., Zhuang, L., and Dufau, M. L. (1996) J. Biol. Chem. 271, 10242-10246). The three promoter regions were co-localized to the rat chromosomal locus 2ql6, in the order 5'-PIII-PI-PII-3'. To investigate the mechanisms of gonad-specific utilization of PI, the promoter domain, regulatory cis-elements, and trans-factors were identified in gonadal cells. The promoter domain localized to the 152-base pair 5' of the transcriptional start site at -549 is highly active in gonadal cells but has minimal activity in hepatoma cells. It contains a steroidogenic factor 1 (SF-1) element (-668) that binds the SF-1 protein of nuclear extracts from gonadal cells and is essential for promoter activation. A CCAAT box (-623) contributes minimally to basal activity in the absence of the SF-1 element, and two adjacent TATA-like sequences act as inhibitory elements. Thus, PI belongs to a class of TATA-less/non-initiator gene promoters. These findings demonstrate an essential role for SF-1 in transcriptional activation of promoter I of the prolactin receptor gene, which may explain the tissue-specific expression of PI in the gonads but not in the liver and the mammary gland.