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In adult humans, the follicle-stimulating hormone (FSH) receptor is expressed only in the granulosa cells of the ovary and the Sertoli cells of the testis. It is minimally expressed by the endothelial cells of gonadal blood vessels.We used immunohistochemical and immunoblotting techniques involving four separate FSH-receptor-specific monoclonal antibodies that recognize different FSH receptor epitopes and in situ hybridization to detect FSH receptor in tissue samples from patients with a wide range of tumors. Immunoelectron microscopy was used to detect FSH receptor in mouse tumors.In all 1336 patients examined, FSH receptor was expressed by endothelial cells in tumors of all grades, including early T1 tumors. The tumors were located in the prostate, breast, colon, pancreas, urinary bladder, kidney, lung, liver, stomach, testis, and ovary. In specimens obtained during surgery performed to remove tumors, the FSH receptor was not expressed in the normal tissues located more than 10 mm from the tumors. The tumor lymphatic vessels did not express FSH receptor. The endothelial cells that expressed FSH receptor were located at the periphery of the tumors in a layer that was approximately 10 mm thick; this layer extended both into and outside of the tumor. Immunoelectron microscopy in mice with xenograft tumors, after perfusion with anti–FSH-receptor antibodies coupled to colloidal gold, showed that the FSH receptor is exposed on the luminal endothelial surface and can bind and internalize circulating ligands.FSH receptor is selectively expressed on the surface of the blood vessels of a wide range of tumors. (Funded by INSERM.).

Thyroid-stimulating hormone receptor (TSHR) consists of a hormone-binding extracellular subunit and a seven-transmembrane spanning subunit that interacts with the G proteins G(alphas) and G(alphaq). The two subunits, generated by proteolytic cleavage of a single polypeptide chain, are held together by disulphide bridges. The receptor is completely cleaved in thyroid tissue, while in cultured cells (thyrocytes and non-thyroid cells) the cleaved and uncleaved forms coexist. The reasons for these divergent data are not understood. Here we provide an explanation by showing that cleavage depends on cell-cell contacts. An almost complete cleavage was observed in confluent cells, while in sparse cells most of the receptor was in the uncleaved form. We also show that coupling of TSHR to G(alphaq) (as measured by inositolphosphate generation) is markedly reduced when the receptor is not cleaved. In contrast, coupling to G(alphas) [as measured by cyclic adenosine 3',5'-monophosphate (cAMP) synthesis] is unaffected by cleavage of the receptor. These results suggest that the cell-cell contacts are necessary for cleavage of the receptor, which acts as a regulatory step in inositolphosphate production via phospholipase C activation. The latter observation was confirmed using cells that express the uncleavable mutant TSHR-delta50-NET, for which the TSH-stimulated inositolphosphate production was completely abolished.

Accumulated data suggest that endothelial cells express specific receptors for several peptide and (glyco)protein hormones that may transport hormones across the cell to be delivered to the interstitial fluid and tissue target cells. Surprisingly, very little information is available on the actual endothelial organelles involved in this cellular process. In the present study the transfer of follicle-stimulating hormone (FSH) through the endothelial barrier of rat testes was examined by analysing the binding and transport of gold-tagged recombinant human (rh)FSH under various conditions using electron microscopy. At 4 degrees C the probe bound specifically to the luminal surface of the endothelial cells without internalization. The use of 125I-rhFSH, which allows precise quantitation of the binding, confirmed the specificity of hormone interaction with the testicular microvasculature. At 37 degrees C the hormone was internalized via coated pits and vesicles into an extensive subluminal tubulo-vesicular compartment and was transported across the endothelium via a system of tubules and vesicles. Moreover, monoclonal antibodies against the FSH receptor ectodomain coupled to colloidal gold followed the same route. In contrast, a non specific, fluid-phase uptake via caveolae was observed for a major plasma protein - rat serum albumin and a fluid-phase tracer - peroxidase. These results suggest that FSH transcytosis across the testicular endothelial barrier is receptor-mediated and involves luminal uptake via coated pits/vesicles, sorting at the level of luminal early endosomes, and transcellular transport through transcytotic tubulo-vesicular organelles. Similar receptor-mediated pathways are likely to be involved in the physiological functioning of a number of other protein and peptide hormones that must translocate specifically from blood to the target cells.

The thyrotropin (TSH) and follicle-stimulating hormone (FSH) receptors are present mainly on the basolateral cell surface in the thyroid gland and in Sertoli cells, whereas in ovarian and in testicular cells, the luteinizing hormone (LH) receptors are distributed throughout the cell surface. When expressed in Madin-Darby canine kidney (MDCK) cells, all three receptors accumulated at the basolateral cell surface showing that they carry the corresponding targeting signals. The receptors were directly delivered to the basolateral surface of the MDCK cells. A minor fraction of the gonadotropin receptors but not of TSH receptors was secondarily targeted to the apical surface through transcytosis. The mechanisms of basolateral targeting and transcytosis were analyzed using the FSH receptor as a model. Both were insensitive to brefeldin A and pertussis toxin. Gs activation by AlF4− and cholera toxin provoked a marked enhancement of FSH receptor transcytosis. The population of Gs proteins involved in this mechanism was different from that involved in signal transduction since neither FSH nor forskolin mimicked the effects of AlF4− and cholera toxin. Gs activation provoked a similar effect on LH receptor distribution in MDCK cells, whereas it did not modify the compartmentalization of the TSH receptor. Hormone-specific transcytosis was observed in MDCK cells expressing the gonadotropin (FSH and LH) receptors and was increased after cholera toxin administration.

The thyrotropin (TSH) receptor in human thyroid glands has been shown to be cleaved into an extracellular alpha subunit and a transmembrane beta subunit held together by disulfide bridges. An excess of the latter component relative to the former suggested the shedding of the ectodomain. Indeed we observed such a shedding in cultures of human thyrocytes and permanently transfected L or Chinese hamster ovary cells. The shedding was increased by inhibitors of endocytosis, recycling, and lysosomal degradation, suggesting that it was dependent on receptor residency at the cell surface. It was slightly increased by TSH and phorbol esters, whereas forskolin and 8-bromo-cyclic AMP were without effect. Decreasing the serum concentration in cell culture medium enhanced the shedding by an unknown mechanism. The shedding of the TSH receptor alpha domain is the consequence of two events: cleavage of the receptor into alpha and beta subunits and reduction of the disulfide bridge(s). The complete inhibition of soluble TSH receptor shedding by the specific inhibitor BB-2116 indicated that the cleavage reaction is catalyzed probably at the cell surface by a matrix metalloprotease. This shedding mechanism may be responsible for the presence of soluble TSH receptor alpha subunit in human circulation.

The cloning and sequencing of porcine lutropin/choriogonadotropin (LH/hCG) receptor messenger RNAs have shown the presence of a full-length receptor (pLHR-A) and of shorter variants lacking either the transmembrane and the intracellular domains (pLHR-B and pLHR-C) or only the transmembrane domain (pLHR-D). Moreover, immunoblotting of testicular membrane extracts has detected 85-, 68-, and 45-48-kDa proteins reacting with antireceptor antibodies. Transfection experiments were performed to assign the protein species to the various messenger RNAs and to study the function of the various receptor species. COS-7 and L-cells transfected with an expression vector encoding full-length receptor pLHR-A yielded a protein of apparent molecular mass of 105 kDa. This corresponded to the complete receptor which had undergone a different glycosylation pattern to that found in testis, since after digestion with peptide N-glycosidase F both the 105-kDa COS-7 protein and the 85-kDa testicular glycoprotein yielded a holoprotein of approximately 63 kDa. Transfection with pLHR-A also yielded a high proportion of the 68-kDa glycoprotein which was shown by digestion with endoglycosidase H to be a high-mannose precursor of the full-length receptor. The existence of a large pool of precursor species in both transfected cells and Leydig cells evokes possible physiological regulations at the level of receptor maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Our present knowledge of the lutropin (LH/hCG) receptor structure derives from deductions made from its amino acid sequence as established by studying the cDNA. To obtain direct experimental information, luteinizing hormone (LH) receptor expressed in L cells was immunopurified in sufficient amounts to warrant analysis by mass spectrometry and microsequencing. The mature receptor, complexed to human chorionic gonadotropin (hCG), was purified by using monoclonal antibodies recognizing the hormone, whereas the mannose-rich non-hormone-binding precursor was purified by use of antireceptor antibodies. Determination of the N-terminus showed that (2)/(3) of protein molecules started at Thr24 whereas (1)/(3) started at Ala28. All these molecules bound hCG, suggesting that the most N-terminal region of the receptor does not participate in hormone binding. Six N-glycosylation sites have been predicted from the amino acid sequence. One of them (Asn299) was found to be nonglycosylated in both the precursor and the mature protein. The most heavily glycosylated residue was Asn291, followed by Asn195 and Asn99. These three sites accounted for 82% and 97% of carbohydrate moieties in the mature receptor and in the mannose-rich precursor, respectively. The presence of some receptor molecules nonglycosylated at sites 99, 174, and 195 in hormone-receptor complexes dismisses a direct role of these glycosylation sites in hormone binding or in the correct folding of the protein. The mature carbohydrate chains were homogeneous at position 174, 195, and 313 (absence of Golgi mannosidase II activity at positions 174 and 313, absence of GlcNAc tranferases III and IV activity at position 195). Heterologous carbohydrates were present at sites 99 and 291. The latter, which is highly variable in carbohydrate chains, is unlikely to participate in a direct interaction with hormone. Site 313 thus remains as the main candidate for a role in hormone binding.

Progesterone receptor from rabbit uterine cytosol was purified to a specific activity of approximately 2 nmol of bound hormone per mg of protein. A goat was immunized with this preparation and, after two injections of 0.7-0.8 nmol, yielded antireceptor antibodies. The antiserum reacted with both cytosolic and nuclear rabbit progesterone receptor and also with progesterone receptor from other rabbit tissues (vagina and pituitary). A crossreaction was observed with progesterone receptors from other mammalian, especially human, tissues (cytosolic receptor from rat and guinea pig uterus, cytosolic receptor from human breast cancer, and nuclear receptor from human endometrium). On the contrary, there was no interaction with a nonmammalian receptor (chicken oviduct progesterone receptor). The antibodies did not crossreact with other rabbit steroid receptors (uterine estradiol receptor and liver glucocorticoid receptor) or with nonreceptor progesterone-binding proteins (transcortin from plasma and uteroglobin from uterine fluid).

Nuclear receptors for both estradiol and progesterone were present in twofold higher concentrations in implantation sites than in nonimplantation regions of the endometrium of 6-day pregnant rats. Decidualization in the absence of an embryo was not accompanied by a similar increase in the concentration of nuclear receptors. Moreover, this difference in receptor distribution between the implantation and nonimplantation areas persisted when a major part of the maternal supply of sex steroids was suppressed by ovariectomy on day 5 of pregnancy. These results support the hypothesis that steroids originating from the embryo affect the endometrial implantation site.

Estrogens play an important role in the genesis of experimental and human breast cancers1,2. Once established, the tumors are frequently hormone-dependent, i.e. they regress or stop growing if deprived of estrogen3. in humans, about one-third of advanced breast cancers are thus susceptible to remission with endocrine therapy4. Criteria for the selection of patients that will respond to hormonal treatment have been the subject of intense study during the last 2 decades5. On the molecular level this had led to the development of analytical tools for the study of steroid receptors and steroid-induced proteins.

A method is described to map contiguous epitopes recognized by monoclonal antibodies in the case when the cDNA for a protein has been cloned. The cDNA is inserted into an expression vector allowing its acellular transcription, followed by the translation of the resulting messenger RNA. C-terminally truncated species of the protein are either generated by cutting the cDNA with restriction enzymes or arise spontaneously through stops occurring during translation of the mRNA. If necessary, progressive digestion by Bal31 of the cDNA can be used to produce an array of polypeptides having different C-terminal lengths. Immunoprecipitation then allows determination of the shortest protein recognized by the monoclonal antibody and thus to define its site of action. This method has been applied to the study of a group of selected monoclonal antibodies among the 59 that have been prepared against the rabbit progesterone receptor. Four immunogenic domains were identified lying between amino acids 1-60, 101-110, 295-325 and 370-396. There were no antibodies directed against the DNA-binding or the steroid-binding regions of the receptor. This is probably due to the high degree of amino acid sequence conservation in these domains, observed when comparing receptors from different species. The antibodies cross-reacting with highest affinity for the human receptor interact with the first immunogenic domain (amino acids 1-60). The 79-kDa form (‘subunit A’) of the receptor was shown to lack the two more N-terminally localized immunogenic domains (amino acids 1-60 and 101-110). The 65-kDa form lacked, in addition, the domain localized between amino acids 295 and 325. These two forms of the receptor thus correspond to deletions of the N-terminal part of the protein.