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.

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.

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.

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.

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.

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.

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.

The genomic structure of the rat corticotropin releasing factor receptor. A member of the class II G protein-coupled receptors.

Isolation and structural characterization of the rat corticotropin releasing factor receptor (CRFR) gene was performed to determine the exon/intron organization of the coding region and the potential for splice variants. The CRFR gene contains 13 exons and 12 introns, and the positions of the exon/intron junctions are similar to those of other Class II G protein-coupled receptor genes including the parathyroid hormone and glucagon receptors. The promoter resides within 593 base pairs of the initiation codon and the major transcriptional start site at nucleotide -238. This domain does not possess a TATA box but contains multiple Sp1 and AP-2 sites upstream and downstream of the major transcriptional start site. Intron junctions were identified in the extracellular, transmembrane (TM), and cytoplasmic (C) domains of the CRFR, giving the potential for differential signal transduction by splice variants. CRFR cDNAs derived from rat Leydig cell mRNA included the pituitary Form A, which spans exons 1-13, and two splice variants with deletion of exon 3 or exons 7, 11, and 12. An evolutionary link between the intronless TM/C module of the glycoprotein hormone receptors and the intron-containing TM/C module of the CRFR is suggested by the common position of the luteinizing hormone receptor Form D alternate acceptor splice site and the CRFR intron 12.

Structure and regulation of the luteinizing hormone receptor gene.

Studies of the mechanisms controlling the expression of the rat luteinizing hormone receptor gene were pursued by characterization of the gene structure and identification of regulatory protein binding domains in the 5'-non-coding region of the gene and of 3' non-coding functional domains responsible for generation of the major mRNA forms. The coding region of the rat LHR gene contains 10 introns and 11 exons, of which the first 10 exons comprise the hormone binding extracellular domain and exon 11, the seven transmembrane/G protein coupling module. Several alternative spliced variants of the LHR were identified that conform to deletions of complete and/or partial exons. Within the 6.2 kb of the 3'-non-coding region, two functional LHR pA domains (H1) and (H2) produce two sets of major mRNA transcripts, each coding for both holoreceptor and the form B splice variant. The H1 pA domain is unique to LHR and may represent a recombinant insertion domain. The functional efficiency of each pA domain is related to the specific pA signals, distal downstream elements, and tissue-specific factors. A TATA-less promoter region is present within the 173 bp 5'flanking region of the gene, with Initiator (Inr) elements at transcriptional start sites. Transcription is dependent on the binding of the Sp1 protein at two Sp1 domains that each contribute equally to transcript initiation. Promoter activity is regulated by at least three additional DNA domains, R (-1266 to -1307 bp), C-box (-42 to -73 bp) and M1 (-24 to -42 bp) that bind multiple trans-factors in a tissue-specific manner. Basal promoter activity is enhanced by a functional M1 domain in LHR-expressing mouse Leydig tumor cells (MLTC) but not in non-expressing CHO cells. C-box binding factors either inhibit promoter activity or block inhibition through overlapping but not identical DNA binding domains that carry AP-2 and NF-1 elements. Removal of the AP-2 element within the C-box results in MLTC-specific transcriptional activation that may involve an MTLC M1/C-box interaction. In addition, competition for C-box factors by an upstream regulatory element (R) that is only inhibitory in CHO cells, indicates that both C-box binding factors compete for this upstream (R) domain in a tissue-specific manner. Competition between the inhibitory and neutral DNA binding factors within both upstream (R) and promoter domains (C-box) could provide a mechanism for the control of LH receptor gene expression in gonadal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of diverse functional elements in the upstream Sp1 domain of the rat luteinizing hormone receptor gene promoter.

Transcription of the luteinizing hormone receptor gene is dependent on Sp1-induced promoter activation from two Sp1 binding domains (Sp1(2), and Sp1(4)) within the 173-base pair promoter. Of the two Sp1 binding domains, the canonical GC box (GGGCGG) was determined by mutation to be the binding element for only the Sp1(2) domain. The Sp1 binding element within the Sp1(4) domain was identified by mutation and immunological/competition studies as the 5'-GGG GTG GGG that conforms to a Zif-268 like three zinc finger binding domain, rather than the canonical 3' Sp1(4) GC box (GGGCGG). The guanines in the third trinucleotide (GGG GTG GGG) were not required for Sp1 binding, although they increased binding affinity. Non-Sp1 protein(s) bind the 3' Sp1(4) GC box, and by themselves exhibit transcriptional activity. Tissue specific differences were localized to this non-Sp1 binding domain, which functionally substituted for the downstream activating M1 regulatory domain in non-expressing but not in expressing cells. Mutations of both non-Sp1 and M1 domains were required for inhibition of promoter activity in constructs that retained the Sp1 binding elements in non-expressing cells, indicating that together these domains may play a role in regulation of luteinizing hormone receptor gene expression.

Transcriptional protein binding domains governing basal expression of the rat luteinizing hormone receptor gene.

The functional importance of specific protein binding domains on transcription within the GC-rich 173-base pair promoter of the luteinizing hormone receptor gene was studied by mutagenesis and gel retardation analysis. Transcription was dependent on the presence of two Sp1 elements in the promoter domain of transfected expressing mouse Leydig tumor cells (mLTC) and nonexpressing Chinese hamster ovary cells. Mutation of two protein binding domains located downstream of the Sp1 elements (M1 and C-box) revealed tissue-specific regulation of promoter activity by each domain. Also, gel retardation studies indicated the presence of multiple trans factors that bind to the C-box and M1 domains. Removal of the AP-2 element from the C-box resulted in mLTC-specific transcriptional activation that may involve an M1/C-box interaction. In addition, competition by overlapping NF-1 and AP-2 elements was demonstrable in both the C-box and upstream R domain for separate trans factors that exhibit neutral or inhibitory functions, respectively. Competition between the inhibitory and neutral DNA binding factors within both upstream and promoter domains may be responsible for a mechanism that controls the on/off state of luteinizing hormone receptor gene expression in gonadal cells. These studies reveal a complex pattern of transcriptional regulation that may reflect targeted mechanisms for the control of luteinizing hormone receptor gene expression.

Promoter and regulatory regions of the rat luteinizing hormone receptor gene.

The region of transcriptional activity in the rat luteinizing hormone receptor gene was localized to the 5'-flanking 173-base pair (bp) sequence in transfected mouse Leydig tumor cells and non-expressing Chinese hamster ovarian (CHO) and HeLa cells. Repression of activity in all cell types was induced by at least two domains located between -174 and -990 bp. An additional inhibitory region between -1237 and -2056 bp observed only in CHO and HeLa cells may contribute to the absence of receptor expression in these cells. Analysis of the 173-bp region revealed a promoter domain between -1 and -137 bp containing no TATA or CAAT boxes, but with three SP1 sites and three initiator elements located at transcriptional start sites -14, -19, and -33. Gel retardation studies showed a protein-binding DNA domain about 30 bp upstream of the transcriptional start sites and an adjacent domain containing an SP1 element that are required for maximal activity in all cell types and may play a role in basal transcription. A second promoter domain (-120 to -173 bp) with a protein-binding SP1 element had minor activity in Leydig cells but was prominent in CHO and HeLa cells. Leydig cell-specific DNA-binding protein(s) for each of the promoter domains were detected and may be of importance in transcriptional regulation. The control of gene transcription by differential activation of these promoter domains could be involved in hormone-induced regulation of luteinizing hormone receptor expression in the gonads.

Changes in binding activity of luteinizing hormone receptors by site directed mutagenesis of potential glycosylation sites.

Site directed mutagenesis of the rat ovarian luteinizing hormone (LH) receptor cDNA was performed at each of the six potential N-linked glycosylation sites to determine the effect of putative carbohydrate chains on the activity of the membrane receptor. The conversion of Asn173 to Gln resulted in the total loss of hormone binding to the surface of the transfected cell. Mutant receptors synthesized with substitutions at the remaining potential N-linked glycosylation positions of 77, 152, 269, 277 and 291 revealed no significant change in the hormone affinity. However Asn77Gln and Asn152Gln exhibited significant decreases (approximately 80%) in the number of high affinity hormone binding sites. The changes in hormone binding activity upon elimination of the potential glycosylation sites at 77, 152 and 173 indicate the presence of functional carbohydrate chains at these positions in the rat ovarian LH/hCG receptor.

Structural organization of the rat luteinizing hormone (LH) receptor gene.

The luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor gene was isolated from rat liver genomic libraries and spanned at least 75 kilobase pairs from nucleotide -2057 of the 5'-flanking region to the 3'-noncoding end. The structural configuration of the coding region of the LH receptor gene consists of 11 exons separated by 10 introns that are all located within the putative extracellular domain prior to the first transmembrane region. The 5'-noncoding region contains several potential TATA boxes and SP1 promoter binding sites, as well as six palindromic elements, potential intron/exon splice junctions, and two extended open reading frames in frame with the initiation codon. Primer extension studies indicate the presence of multiple transcriptional initiation sites. Truncated forms of the LH/hCG receptor conform to alternative splicing patterns that are consistent either with the deletion of complete exons or alternate acceptor sites within exons. All splice site junctions correspond to known donor and acceptor consensus sequences. Exons 2-8 approximate the regions of the 14 consecutive 20 amino acid repeated motifs present in the LH, thyrotropin-stimulating hormone, and follicle-stimulating hormone receptor cDNAs, with the exception of a six to eight amino acid shift in each motif. Exons 1, 9, 10, and 11, do not conform to this repetitive intronic motif. These exons, however, contain important structural elements including the conserved cysteines (exons 1, 9, 11), the soybean lectin motif (exon 9), the seven-transmembrane domain with cytoplasmic G protein coupling elements (exon 11), and three putative N-linked glycosylation sites (exon 10), consistent with preservation of significant functional domains within single exons. Exon 10 and the beginning of exon 11 along with the lectin motif are unique to the LH receptor and may be involved in specific association with the hormonal ligand. The homologous regions with other members of the glycoprotein receptor family encoded by exons 2-8, and the common amino acid motif that contains 3 conserved cysteines immediately prior to the first transmembrane region, may be involved in common hormonal interactions and in coupling functions, respectively.

Intronic nature of the rat luteinizing hormone receptor gene defines a soluble receptor subspecies with hormone binding activity.

A rat testicular luteinizing hormone (LH) receptor cDNA containing a 266-base pair deletion resulting in the omission of the 1st transmembrane region and truncation of the open reading frame was isolated using a rat ovarian LH receptor cDNA probe. Comparison of this clone with a restriction fragment from the LH receptor genomic DNA revealed potential alternative splice sites following the consensus sequence TTXCAG that is present at an intron acceptor splice site and also within the next exon, accounting for the specific deletion mutation observed in this cDNA. Expression of the testicular cDNA in COS1 cells resulted in synthesis and secretion of a soluble binding protein with high affinity and specificity for LH and human chorionic gonadotropin. These studies have demonstrated that the LH receptor gene contains intron(s) within the region coding for the extracellular domain of the molecule, which determine the nature and generation of LH receptor isoforms. Expression of the soluble form of the LH receptor has indicated that the amino-terminal extracellular region plays a major role in gonadotropin binding. These features of the LH receptor are distinct from those of most other G protein-coupled receptors, which are intronless and contain their binding sites within the transmembrane region rather than the extracellular domain.

Hormonal regulation of LH receptor mRNA and expression in the rat ovary.

Agonist-induced changes in expression and mRNA levels of luteinizing hormone (LH) receptors were compared during stimulation of ovarian follicular maturation and luteinization by gonadotropic hormones. Three major species of LH receptor mRNA, 5.8, 2.6 and 2.3 kb, were present throughout differentiation and changed similarly, the 5.8 kb species being consistently more abundant than the smaller forms. The increased expression of plasma-membrane LH receptors in preovulatory follicles and luteinized ovaries and their homologous down-regulation during follicular and luteal desensitization were closely correlated with the steady-state receptor mRNA levels. The reappearance of LH receptors following desensitization during the luteal stage was preceded by an increase in mRNA levels. These studies have demonstrated that the expression of LH receptors during follicular maturation, ovulation and desensitization is related to the prevailing levels of receptor mRNA in the ovary.

Isolation and characterization of two novel rat ovarian lactogen receptor cDNA species.

Two novel lactogen receptor cDNA clones (2.1 and 1.2 kb) were isolated from a rat ovarian cDNA library. Nucleotide sequence of the 2.1 kb clone codes for a 610 aa receptor (nonglycosylated mol. wgt. 66,000 D) with an extracellular domain, a transmembrane region and an intracellular domain, and exhibited significant overall similarity with the rat liver receptor (310 aa) and both rabbit mammary and human hepatoma receptors (616 and 622 aa). However, the ovarian lactogen receptor sequence contains a unique cytoplasmic domain of 110 aa and consensus sequences for both a tyrosine phosphorylation site and an ATP/GTP type A binding site, and thus has potential for signal transduction and mitogenic activity. The 1.2 kb clone codes for a truncated binding form of 150 aa that is identical with the ovarian long form over only the first 130 residues, and lacks the transmembrane region. Differences between long and short forms of the ovarian lactogen receptors and the truncated liver species may result from alternative splicing. The prolactin holoreceptor gene(s) has the potential for producing several receptor subtypes that differ in tissue-specific expression, size, compartmentalization and mode of signal transduction, and may subserve the divergent functions of prolactin in its several target cells.