Genomic checkpoints for exon 10 usage in the luteinizing hormone receptor type 1 and type 2.

Alternative splicing is a hallmark of glycoprotein hormone receptor gene regulation, but its molecular mechanism is unknown. The LH receptor (LHR) gene possesses 11 exons, but exon 10 is constitutively skipped in the New World monkey lineage (LHR type 2), whereas it is constitutively spliced in the human (LHR type 1). This study identifies the regulatory elements of exon 10 usage. Sequencing of genomic marmoset DNA revealed that the cryptic LHR exon 10 is highly homologous to exon 10 from other species and displays intact splice sites. Functional studies using a minigene approach excluded the contribution of intronic, marmoset-specific long interspersed nucleotide-1 elements to exon 10 skipping. Sequencing of the genomic regions surrounding exon 10 from several primate lineages, sequence comparisons including the human and mouse LHR gene, revealed the presence of unique nucleotides at 3'-intronic position -19 and -10 and at position +26 within exon 10 of the marmoset LHR. Exon trap experiments and in vitro mutagenesis of these nucleotides resulted in the identification of a composite regulatory element of splicing consisting of cis-acting elements represented by two polypyrimidine tracts and a trans-acting element within exon 10, which affect the secondary RNA structure. Changes within this complex resulted either in constitutive exon inclusion, constitutive skipping, or alternative splicing of exon 10. This work delineates the molecular pathway leading to intronization of exon 10 in the LHR type 2 and reveals, for the first time, the essential function of regulatory and structural elements involved in glycoprotein hormone receptor splicing.

A new subclass of the luteinizing hormone/chorionic gonadotropin receptor lacking exon 10 messenger RNA in the New World monkey (Platyrrhini) lineage.

The luteinizing hormone receptor (LHR) plays an essential role as a mediator of LH and CG action during embryonic sexual differentiation and in gametogenesis. In a hypogonadal male patient, we recently demonstrated that a genomic deletion of exon 10, located in the hinge region of the extracellular domain, results in discrimination of LH and hCG action. In the common marmoset (Calltithrix jacchus), exon 10 of the LHR is naturally missing at the mRNA level. In order to investigate whether this is an isolated species-specific phenomenon, we performed a phylogenetic screening, searching for the presence of LHR exon 10 mRNA in a number of primate species representative for the major lineages of primate evolution. The expressed LHR region encompassing exon 10 was amplified from testicular tissue by RT-PCR, cloned, and sequenced. In addition, we performed Southern blot analysis of the LHR of selected New World and Old World primates. The results revealed that exon 10 mRNA is lacking in the complete New World monkey (Platyrrhini) lineage but is present in both more primitive and more advanced primates. However, exon 10 seems to be present at the genomic level, arguing for a splicing failure possibly due to a genomic mutation or the lack of appropriate splicing factors. Considering that, in the human, LH is far less active than hCG on the LHR lacking exon 10, we addressed the question whether the existence of such a receptor has any consequences on the dual hormone LH/CG system present in Platyrrhini. Using primers specific for the known marmoset CG beta cDNA, we amplified the CG beta subunit cDNA from male common marmoset pituitaries by RT-PCR, while LH beta could not be amplified, suggesting a possible physiological role of pituitary CG in this species. In conclusion, we demonstrated for the first time that the LH mRNA without exon10 is the natural wild-type LHR in the Platyrrhini lineage. We propose that this LHR represents a new subclass of receptors that should be named LHR type II. In addition, the high expression of CG beta in the marmoset pituitary suggests a physiological role of CG in the reproductive function of these primates beyond pregnancy.

[Pituitary glycoprotein hormone receptors]. / Les récepteurs des hormones glycoprotéiques hypophysaires.

Monoclonal antibodies have been raised against the porcine LH receptor and have allowed to clone the corresponding messenger RNA from testicular cells. The stricture of the LH receptor has been determined. It shows similarities but also differences with other G protein coupled receptors. Specially a large extracellular domain is specific of that new family of receptors. Variant forms of the LH receptor generated by alternative splicing and lacking transmembrane domain have been isolated. Immunochemical and immunocytochemical studies have been performed. Three different forms of the LH receptor are physiologically expressed: a mature 85kDa transmembrane species, a 68 kDa high mannose containing species corresponding to a precursor which accumulates inside the cells, and truncated 45-48kDa molecular weight species corresponding to the variant messenger RNAs identified during the cloning of the receptor. A novel zonation of the ovary has been described by immunocytochemical studies. Cross hybridization with the LH receptor clone allowed to isolate the related TSH receptor from human thyroid tissue. The human LH and FSH receptor genes have been localized to chromosome 2p21 and the TSH receptor gene to chromosome 14q31. The genes are very large (> 60 kbp) and have introns only within the 5' part encoding the extracellular domain of the receptor. Immunoelectron microscopic studies performed in Leydig cells and in stably transfected L cells have allowed to study intracellular traffic of the LH receptor. The same approach was used to study the transendothelial transfer of hCG in testicular microvasculature.

[LH receptors. A new family of G-protein receptors]. / Récepteur de la LH. Une nouvelle famille de récepteurs couplés à des protéines G.

Monoclonal antibodies have been raised against porcine LH receptor and allowed to clone the corresponding messenger RNA from testicular cells. The structure of the LH receptor have been determined. It shows similarities but also differences to other G protein coupled receptors. In particular a large extracellular domain is specific for that family of receptors. Variants forms of the LH receptor generated by alternative splicing and lacking transmembrane domains have been isolated. Immunochemical and immunocytochemical studies have been performed. Three different forms of the LH receptor are physiologically expressed: a mature 85 kDa transmembrane species, a 68 kDa high mannose containing species corresponding to a precursor which accumulate inside the cells, and truncated 45-48 kDa molecular weight species corresponding to the variant messenger RNAs identified during the cloning of the receptor. A novel zonation of the ovary has been described by immunocytochemical studies. Cross hybridisation with the LH receptor clone allowed to isolate the related human TSH receptor from thyroïds. The human LH and FSH receptor genes have been localized to chromosome 2p21 and the TSH receptor gene to chromosome 14q31. The genes are very large and have introns only within their 5' part corresponding to the extracellular domain of the receptor.

[LH and TSH receptors. A new family of G protein-coupled receptors]. / Récepteurs de la LH et de la TSH. Une nouvelle famille de récepteurs couplés à des protéines G.

Monoclonal antibodies have been raised against porcine LH receptor and allowed to clone the corresponding messenger RNA from testicular cells. Cross hybridisation with the LH receptor clone allowed to isolate a clone corresponding to the human TSH receptor from thyroids. The structure of both receptors have been determined. They show similarities but also differences to other G protein coupled receptors. In particular a large extracellular domain is specific of that new family of receptors. Variant forms of the LH receptor lacking transmembrane domains have been isolated. The obtention of monoclonal antibodies against both receptors allowed immunochemical and immunocytochemical studies to be performed. The human LH receptor gene have been localized to chromosome 2p21 and TSH receptor gene to chromosome 14q31. The complete organisation of the human TSH receptor gene has been determined.