Research resource: novel structural insights bridge gaps in glycoprotein hormone receptor analyses.

The first version of a glycoprotein hormone receptor (GPHR) information resource was designed to link functional with structural GPHR information, in order to support sequence-structure-function analysis of the LH, FSH, and TSH receptors (http://ssfa-gphr.de). However, structural information on a binding- and signaling-sensitive extracellular fragment (∼100 residues), the hinge region, had been lacking. A new FSHR crystal structure of the hormone-bound extracellular domain has recently been solved. The structure comprises the leucine-rich repeat domain and most parts of the hinge region. We have not only integrated the new FSHR/FSH structure and the derived homology models of TSHR/TSH, LHCGR/CG, and LHCGR/LH into our web-based information resource, but have additionally provided novel tools to analyze the advanced structural features, with the common characteristics and distinctions between GPHRs, in a more precise manner. The hinge region with its second hormone-binding site allows us to assign functional data to the new structural features between hormone and receptor, such as binding details of a sulfated tyrosine (conserved throughout the GPHRs) extending into a pocket of the hormone. We have also implemented a protein interface analysis tool that enables the identification and visualization of extracellular contact points between interaction partners. This provides a starting point for comparing the binding patterns of GPHRs. Together with the mutagenesis data stored in the database, this will help to decipher the essential residues for ligand recognition and the molecular mechanisms of signal transduction, extending from the extracellular hormone-binding site toward the intracellular G protein-binding sites.

[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.

Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells.

Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.

Differences in properties of the sheep testicular LH and FSH receptors.

Differences in binding and structural properties of ovine testicular FSH and LH receptors were investigated. The ovine FSH receptor did not discriminate between FSH of different species, although equine FSH was more reactive. In the same tissue, however, the LH receptor showed marked preference for ovine and bovine LH, reacting very weakly with other preparations of pituitary LH. Human chorionic gonadotrophin also reacted partly with the ovine LH receptor at 25 degrees C. However, at 4 degrees C, the optimum temperature for binding of the LH receptor to its homologous hormone, the receptor displayed no recognition for chorionic gonadotrophin preparations. Affinity cross-linking studies with ovine testicular membrane suggested that the ovine FSH receptor has an Mr of 70,000, which is very similar to that observed in the porcine ovary. The Mr of the ovine LH receptor was estimated to be 150,000, which is different from those of other mammalian species, including those that have been cloned. The data suggest that the binding and structural properties of the ovine FSH receptor are similar to those of other mammalian FSH receptors, whereas the ovine LH receptor appears to differ from other mammalian LH receptors in having a different Mr and in being more stringent in its requirement for pituitary LH.

Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity.

The lutropin-choriogonadotropin (LH/CG) receptor is a cell surface receptor comprised of two domains of roughly equivalent size. The amino-terminal half of the receptor is relatively hydrophilic and is located extracellularly, whereas the carboxyl-terminal half of the receptor shares amino acid homology with other receptors that couple to G proteins and is similarly thought to span the plasma membrane seven times, ending with a relatively short carboxyl-terminal tail. In order to test the role of the extracellular domain in binding hormone, we constructed a mutated rat luteal LH/CG receptor cDNA (termed pCLHR-D2), which encodes for only the extracellular domain, and used it to transiently transfect human kidney 293 cells. Here we report that the expressed extracellular domain of the LH/CG receptor is capable of binding human CG with a high affinity, comparable with that of the full-length receptor. Thus, not only is the extracellular domain of the glycoprotein hormone receptors involved in binding hormone, but it alone is capable of conferring high affinity binding. Unexpectedly, it was also found that this truncated receptor is not secreted into the culture media but remains trapped within the cells.

Steroidogenic enzyme activities, morphology, and receptor studies of a testicular adrenal rest in a patient with congenital adrenal hyperplasia.

Steroid-secreting tumors of the testis have generally been considered to be of Leydig cell origin. Testicular tumors in patients with congenital adrenal hyperplasia have been thought to be adrenal rests, but no conclusive evidence supporting the hypothesis has been presented. We report a morphological and biochemical analysis of a patient with 21-hydroxylase deficiency who developed bilateral nodular hyperplasia of steroid-secreting tissue within the testis, despite suppression therapy with both exogenous glucocorticoids and testosterone. The tissue was formed of confluent nodules of homogenous cells. Electron microscopy showed the cells to have abundant smooth endoplasmic reticulum, well developed Golgi apparatus, and mitochondria with predominantly tubular cristae, features characteristic of steroid-secreting cells of adrenocortical origin. Crystals of Reinke were not observed. Functional studies in vivo showed a marked response to ACTH infusion, with 17-hydroxyprogesterone rising from 56 to 13,500 ng/mL, cortisol from less than 2 to 19 micrograms/dL, and testosterone from 369 to 629 ng/dL, with an attendant increase in testicular size and pain over 48 h. Receptor studies in vitro revealed no gonadotropin receptors, but abundant angiotensin-II receptors. Enzyme activity analysis in vitro showed undetectable 21-hydroxylase activity and an enzyme profile consistent with adrenocortical cells rather than Leydig cells. Based on these morphological and biochemical findings, we conclude that the nodular steroidogenic tissue that replaced this patient's testes was of adrenal origin. The study documents for the first time the development of adrenocortical tumors from adrenal rest tissue within the testis.