The unique exon 10 of the human luteinizing hormone receptor is necessary for expression of the receptor protein at the plasma membrane in the human luteinizing hormone receptor, but deleterious when inserted into the human follicle-stimulating hormone receptor

The LH receptor (LHR) is a member of the family of G protein-coupled seven-times plasma membrane transversing receptors. Its gene consists of 11 exons, the last one encoding the transmembrane and intracellular domains of the receptor. The FSHR, and its gene, resemble structurally those of the LHR, with the exception that the sequences corresponding to exon 10 in LHR are missing in FSHR, which is thus encoded by a total of ten exons. Our recent studies on the marmoset monkey testis LHR cDNA indicated that an 81 bp nucleotide sequence, encoding the complete exon 10 of the LHR gene in other mammalian species, is absent in this species without affecting the LHR function. To study further the role of the exon 10 encoded sequences of the LHR in the gonadotropin receptor function, a deletion of exon 10 from the human LHR (hLHdeltaexon10R), and a chimeric hFSHR with exon 10 from hLHR inserted (hFSHLHexon10R), were constructed in expression vectors. The results presented here demonstrate that 293 cells transfected with the hLHdeltaexon10R display a decrease in the proportion of the receptor binding at the cell surface, compared with cells transfected with wild-type hLHR. However, the cells expressing hLHdeltaexon10R showed similar high affinity binding of [125I]iodo-hCG as those transfected with wild-type hLHR, in either intact cells or their detergent extracts. In addition, cells expressing the hLHdeltaexon10R and wild-type hLHR displayed similar dose-response of cAMP production to hCG stimulation. Cells transfected with chimeric hFSHLHexon10R showed barely detectable [125I]iodo-FSH binding in intact cells compared with those transfected with wild-type hFSHR. The FSH binding detected in cellular detergent extracts displayed 10-fold lower binding activity than wild-type receptors, in spite of similar level of immunoreactive FSHR protein expression in the transfected cells. The hFSHLHexon10R had a modest 5-fold lower binding affinity for FSH as compared with wild-type hFSHR. In conclusion, the present study indicates that the sequences encoding exon 10 of the hLHR are essential for the LHR expression at the plasma membrane, but deleterious for function if inserted into the hFSHR.     

Localization of human follicle-stimulating hormone in the testis

Localization of the follicle-stimulating hormone (FSH) molecule and its receptor (FSHR), as well as the role of FSH in Sertoli cell mitosis and maturation, has been demonstrated by several investigators in human and murine testis by detecting the localization of anti-FSH antibodies or [(131)I]-labeled FSH and by detecting FSH receptor (FSHR) mRNA by in situ hybridization, or FSHR by anti-FSHR antibodies. The presence of FSH in germinal cells is controversial or, in humans, excluded. We have investigated the distribution of the human FSH molecule and its receptor in human and mouse testicular cells under different experimental conditions, at the submicroscopical level, by using a better antigenicity conservative procedure. Thus, the distribution of FSH and of the messenger RNA for its receptor in Sertoli cells has now been clarified. In germinal cells, our observations demonstrate the presence of FSH and the FSHR mRNA: the first on the plasma membrane and in endocytotic vesicles, and the second scattered in the cytoplasm. The cells presenting the higher amount of positivity ranged from spermatogonia to spermatocytes, including round spermatids. Penetration was by the endocytosis via membrane vesicles in which the FSHR is present, whereas its messenger is largely present in the cytoplasm and is responsible for the binding and subsequent internalization of the FSH molecule. As a control, human FSH was administered in vitro to the Y1 mouse cell line, which was stably transfected with cDNA for FSHR and devoid of endogenous FSH. The FSH molecule has been localized by monoclonal antibodies on plasma membranes and vesicles, and the FSHR mRNA was found scattered in the cytoplasm after in situ hybridization. We can now conclude that FSH is present in Sertoli cells and in round germinal cells, both expressing the FSHR. FSH penetrates in a similar way in both kinds of cells via endocytosis, and is therefore subsequently localized in the same membranous organelles.     

Design, synthesis, and biological activities of potent and selective somatostatin analogues incorporating novel peptoid residues

We report the synthesis, bioactivity, and structure-activity relationship studies of compounds related to the Merck cyclic hexapeptide c[Pro6-Phe7-d-Trp8-Lys9-Thr10-Phe11], L-363,301 (the numbering in the sequence refers to the position of the residues in native somatostatin). The Pro residue in this compound is replaced with arylalkyl peptoid residues. We present a novel approach utilizing beta-methyl chiral substitutions to constrain the peptoid side-chain conformation. Our studies led to molecules which show potent binding and increased selectivity to the hsst2 receptor (weaker binding to the hsst3 and hsst5 receptors compared to L-363, 301). In vivo, these peptoid analogues selectively inhibit the release of growth hormone but have no effect on the inhibition of insulin. The biological assays which include binding to five recombinant human somatostatin receptors carried out in two independent laboratories and in vivo inhibition of growth hormone and insulin provide insight into the relationship between structure and biological activity of somatostatin analogues. Our results have important implications for the study of other peptide hormones and neurotransmitters.     

A novel phenotype related to partial loss of function mutations of the follicle stimulating hormone receptor

A single natural loss of function mutation of the follicle stimulating hormone receptor (FSHR) has been described to date. Present in the Finnish population it markedly impairs receptor function, blocking follicle development at the primary stage and presenting as primary amenorrhea with atrophic ovaries. When Western European women with this phenotype were examined for FSHR mutations the result was negative, suggesting that other etiologies corresponding to this clinical pattern are markedly more frequent. We now describe a novel phenotype related to mutations provoking a partial loss of function of the FSHR. A woman with secondary amenorrhea had very high plasma gonadotropin concentrations (especially FSH), contrasting with normal sized ovaries and antral follicles up to 5 mm at ultrasonography. Histological and immunohistochemical examination of the ovaries showed normal follicular development up to the small antral stage and a disruption at further stages. The patient was found to carry compound heterozygotic mutations of the FSHR gene: Ile160Thr and Arg573Cys substitutions located, respectively, in the extracellular domain and in the third intracellular loop of the receptor. The mutated receptors, when expressed in COS-7 cells, showed partial functional impairment, consistent with the clinical and histological observations: the first mutation impaired cell surface expression and the second altered signal transduction of the receptor. This observation suggests that a limited FSH effect is sufficient to promote follicular growth up to the small antral stage. Further development necessitates strong FSH stimulation. The contrast between very high FSH levels and normal sized ovaries with antral follicles may thus be characteristic of such patients.     

New insights into the role of follicle-stimulating hormone in reproduction

Follicle-stimulating hormone (FSH) has been considered essential for normal ovarian follicular development and spermatogenesis. Although this concept remains valid, the recent reports of defects in genes encoding FSH and its receptor (FSHR) have altered the concepts, particularly with regard to the role of FSH in spermatogenesis. Complete FSH resistance caused by a mutation of the FSHR gene has demonstrated that normal FSH action is an absolute requirement for female fertility, but spermatogenesis and male fertility are possible without FSH action. Thus, while normal ovarian function is critically dependent on normal FSH action, male fertility is relatively independent of this hormone. The finding contradicts the potential of inhibition of FSH secretion or action as a means of male contraception.     

Gonadotropin receptors and the control of gonadal steroidogenesis: physiology and pathology

Over the past few years, knowledge of the structure of gonadotropin receptors and their mode of action has rapidly advanced. The cDNA corresponding to the luteinizeng hormone (LH) receptor (LHR) has been cloned, leading to the identification of a novel family of G-protein-coupled receptors. The follicle stimulating hormone (FSH) receptor (FSHR) was thereafter cloned by cross-hybridization with the LHR. Structure-function relationships have been studied by mutagenesis experiments in several laboratories. The cloning and chromosomal localization to chromosome 2p21 of the two human gonadotropin receptor genes has provided insights into their evolutionary relationships. The LHR and FSHR genes are very large and contain 10 and 11 exons respectively. The obtention of monoclonal antibodies against the receptors resulted in the characterization of the receptor proteins. These antibodies also allowed the study of receptor expression in target cells in physiological and pathological conditions. The internalization of the LHR has been studied by electron microscopy. A mechanism of receptor-mediated transcytosis through the endothelial cells of the testes has been described for the LHR. The polarized expression of receptors has been studied. The cloning of gonadotropin receptor genes has opened the field of genetic study of the receptors. Inactivating mutations of the LHR have been described in Leydig cell agenesis or hypoplasia. Different phenotypes, including complete pseudohermaphroditism, ambiguous genitalia and male phenotype, have been described. In the case of the FSHR, only one mutation has been reported in familial ovarian dysgenesis with primary amenorrhea. Related males have variable alterations of spermatogenesis and fertility. Constitutive mutations of the LHR have been reported in familial testotoxicosis. One similar mutation has also been described for the FSHR. Such mutations may lead to the development of a model of receptor activation.     

Bioassays of gonadotropins based on cloned receptors

Because of the microheterogeneities of gonadotropins, immunoreactive measurements of gonadotropins do not necessarily reflect their bioactivity. Follicle-stimulating hormone (FSH) bioassays have relied on measurement of aromatase activity in primary cultures of immature rat Sertoli cells or rat granulosa cells (GAB assay). Luteinizing hormone (LH) bioassays have relied on measurement of androgen production in primary cultures of rat interstitial testicular cells (RICT) or mouse Leydig cells. Those bioassays are cumbersome because they rely on primary culture and on indirect measurement of estradiol or testosterone by RIAs. The cloning of the cDNAs of FSH and LH receptors has allowed the establishment of cell lines expressing human receptors. The cotransfection of the recombinant gonadotropin receptor with a cAMP reporter gene allows a nonisotopic measurement of gonadotropin bioactivity. Furthermore, patient serum can be tested directly without prior extraction. We and other groups have developed a CHO cell line expressing the human FSH receptor and a luciferase reporter gene (CHO-FSHR). The CHO-FSHR assays is specific for FSH and free of serum interference up to a final concentration of 20%. The clinical sensitivity is 3 IU/l, the interCV 16%, the intraCV 8%. Studies were performed in normal women (n = 11) during the menstrual cycle using the CHO-FSHR cells. The ratio of bioactive to immunoactive FSH (B/I) equals 1.1 +/- 0.04 across the follicular and early luteal phase. During the mid to late luteal phase the mean B/I rises significantly to 1.65 +/- 0.07 (P < 0.001). Gonadotropin bioassays based on cloned receptors have been used to search for immunoglobulins, directed against the FSH or the LH receptors in premature ovarian failure patients. No blocking antibodies were found among the 38 women studied. A recent study of FSH bioactivity in patients with FSH secreting pituitary adenomas shows increased values of the B/I ratio. In summary, cell lines expressing the LH and the FSH human receptors are now available. Those homologous systems enable clinicians to study potential forms of mutated FSH or antibodies directed against gonadotropin receptors. Furthermore, bioassays based on cloned receptors are interesting tools to test anti-LH or anti-FSH molecules mainly in contraceptive research.     

A role for neurotransmitters in early follicular development: induction of functional follicle-stimulating hormone receptors in newly formed follicles of the rat ovary

The initiation of follicular growth in the mammalian ovary is a gonadotropin-independent phenomenon. Although some of the intraovarian signaling molecules that control the later phases of this process have been recently identified, the factors involved in the acquisition of gonadotropin receptors by early growing follicles have not been fully defined. In the rat, development of the ovarian innervation precedes the onset of folliculogenesis and occurs before follicles acquire responsiveness to gonadotropins. Because vasoactive intestinal polypeptide (VIP) and norepinephrine (NE), two of the neurotransmitters contained in ovarian nerves, are present in the ovary before the gland becomes responsive to gonadotropins, we sought to determine if VIP and/or NE are able to act on early follicles to facilitate the process of molecular differentiation that leads to gonadotropin dependency. In vitro exposure of 2-day-old rat ovaries to isoproterenol (ISO), a beta-adrenoreceptor agonist, or VIP, a neurotransmitter contained in both sympathetic and sensory nerves, increased the steady state levels of the messenger RNAs encoding cytochrome P-450 aromatase (P-450arom) and FSH receptors (FSHR) within 8 h of treatment. A similar effect was observed following forskolin-induced activation of cAMP formation. In situ hybridization experiments revealed that both the P-450arom and FSHR hybridization signals were localized to follicles. The increase in FSHR messenger RNA was accompanied by formation of functional receptor molecules, as demonstrated by the ability of FSH to stimulate cAMP formation in ovaries preexposed to either ISO or VIP, but not in untreated ovaries. The stimulatory effect of ISO and VIP on the formation of FSHR coupled to the cAMP generating system was not reproduced by phenylephrine, an alpha-adrenergic agonist, or secretin, a member of the VIP family not recognized by ovarian VIP receptors. Treatment of VIP-primed ovaries with FSH resulted in follicular growth, demonstrating that exposure of the gland to the neurotransmitter led to the formation of a functional complement of FSH receptors. These results suggest that ovarian nerves, acting via neurotransmitters coupled to the cAMP generating system, contribute to the differentiation process by which newly formed primary follicles acquire FSH receptors and responsiveness to FSH. Follicles that begin to grow in more densely innervated ovarian regions, may have a selective advantage over those not exposed to neurotransmitter-activated, cAMP-dependent signals and, thus, may become more rapidly subjected to gonadotropin control.     

Structure-function relationship of follicle-stimulating hormone and its receptor

Follicle stimulating hormone (FSH) is one of the two pituitary gonadotrophins involved in the regulation of gonadal function. Structurally, this gonadotrophin is a heterodimer composed of two non-covalently associated subunits containing several heterogenous oligosaccharide residues which play an important role in both the in-vivo and in-vitro bioactivity of the hormone. Its cognate receptor, which belongs to the superfamily of the G protein-linked cell surface receptors, also displays a high degree of functional and molecular complexity. Studies employing monoclonal antibodies, synthetic peptides and/or site directed mutagenesis, have unveiled some of the multiple structural determinants involved in FSH and FSH receptor function and interaction. Despite their structural complexity, both molecules exhibit a high degree of plasticity and diversity that allows formation of distinct ligand-receptor complexes capable of selectively activating or deactivating a variety of signalling pathways. Knowledge and mapping of the structural determinants and functional epitopes for intra- and extracellular hormone action are of paramount importance not only for a better and more detailed understanding of the molecular basis of FSH action and FSH receptor function but also for the rational design of analogues with predicted properties and effects.     

Structure-function relationship of recombinant follicle stimulating hormone (Puregon)

After separation by means of preparative isoelectrofocusing, the isohormones of a Chinese hamster ovary (CHO)-derived recombinant follicle stimulating hormone (rFSH, Puregon) were characterized with respect to structural and functional features. A carbohydrate analysis revealed that rFSH isohormones with a low isoelectric point (pl) have a high sialic acid/galactose ratio and are rich in tri- and tetra-antennary N-linked carbohydrate chains in comparison with the high pl isohormones. The relative basic isohormones exhibit receptor binding activity and intrinsic bioactivity 2-3-fold higher than the relative acidic isohormones. However, due to their lower clearance rate these acidic isohormones displayed a 20-fold higher in-vivo bioactivity in the rat. A comparison of the isohormone profile of rFSH and urinary FSH (Metrodin) revealed that rFSH contains about 2-fold more basic isohormones with pl > or = 4.7 and 2-fold less acidic isohormones with pl < 4.1. In-vitro studies showed that the receptor binding affinity and intrinsic bioactivity of both FSH preparations are similar. Also the in-vivo efficacy and the pharmacokinetic behaviour of rFSH and urinary FSH in the rat were similar, which is not surprising since both preparations were compared in terms of in-vivo bioactivity calibrated in the rat Steelman-Pohley assay. However, in dogs the bioavailability of rFSH was lower than that of urinary FSH, which is in agreement with the higher percentage of relative basic isohormones in rFSH. This suggests that the pharmacokinetic behaviour of FSH in rats and dogs is different, which is supported by the much longer elimination half-life of rFSH and urinary FSH in dogs (27.9 and 30.4 h respectively) compared with rats (11.4 and 10.4 h respectively) for rFSH and urinary FSH respectively. The observed differences in pharmacokinetic behaviour in dogs and rats indicate that the rat Steelman-Pohley assay might not be a valid model for the prediction of the FSH bioactivity in species other than rat.     

Differential regulation of leucine-rich primary response gene 1 (LRPR1) mRNA expression in rat testis and ovary

In immature rat Sertoli cells, leucine-rich primary response gene 1 (LRPR1) represents a follicle stimulating hormone (FSH)-responsive gene; the function of the encoded protein is not yet known. LRPR1 mRNA expression is up-regulated very rapidly and specifically by FSH, both in cultured Sertoli cells and in vivo in regulation in more detail, in testis and ovary of fetal, immature, and adult rats. In addition, we have studied the expression of FSH receptor (FSHR) mRNA in relation to LRPR1 mRNA expression. In rat testis, LRPR1 mRNA and FSHR mRNA followed a similar expression pattern, during postnatal development and also at different stages of the spermatogenic cycle in the adult rat. Furthermore, after short-term challenge of the FSH signal transduction pathway in intact immature rats by injection with a relatively high dose of FSH, an inverse relationship between LRPR1 mRNA (up-regulation) and FSHR mRNA expression (down-regulation) was observed. Similar studies in the ovary provided completely different results. LRPR1 mRNA in the postnatal ovary is present well before expression of FSHR mRNA can be first detected. In addition, incubation of ovaries of immature rats with FSH or dibutyryl cyclic AMP (dbcAMP) did not result in up-regulation of LRPR1 mRNA expression. During fetal development, the LRPR1 mRNA expression pattern involved many more tissues, in contrast to the relatively tissue-specific expression of LRPR1 mRNA in gonads of 21 day old and adult rats. Moreover, LRPR1 mRNA expression could be detected as early as 12.5 days post-coitum, whereas FSHR mRNA is absent at this stage of fetal development. We concluded that the pronounced regulation of LRPR1 by FSH observed in the immature rat testis does not occur in the ovary. Furthermore, in the ovary LRPR1 mRNA expression does not appear to be dependent on FSH action. Finally, the LRPR1 gene product may play a general role during fetal development.     

A synthetic peptide corresponding to the third cytoplasmic loop (residues 533 to 555) of the testicular follicle-stimulating hormone receptor affects signal transduction in rat testis membranes and in intact cultured rat Sertoli cells

Involvement of the third cytoplasmic (3i) loop (residues 533 to 555) of the rat testicular FSH receptor in the mechanism of FSH signal transduction was examined using light membranes prepared from immature rat testes, monolayer cultures of rat Sertoli cells, and a synthetic peptide strategy. This region of the FSH receptor is structurally related to G protein-activator regions identified in other G protein-coupled receptors. FSHR-(533-555) peptide amide stimulated guanine nucleotide exchange in rat testis light membranes, presumably via its interaction with membrane-associated G protein. The peptide failed to inhibit FSH binding to testis membrane receptors, indicating that the nucleotide exchange effect was not a result of peptide interaction with receptor. When incubated with cultured Sertoli cells from immature rat testes, FSHR-(533-555) peptide amide consistently and significantly inhibited FSH stimulation of cAMP and estradiol biosynthesis, but failed to inhibit forskolin stimulation of each. The peptide effect, therefore, was not due to a direct interaction with adenylyl cyclase. Since FSHR-(533-555) peptide amide did not inhibit FSH binding to membrane receptor, these results imply entry of the peptide into the Sertoli cell, possibly by vesicular internalization or diffusion. Indeed, the inhibitory effects of FSHR-(533-555) peptide amide on FSH-stimulated estradiol biosynthesis were prevented by pretreating Sertoli cells with phenylarsine oxide, an inhibitor of FSH receptor internalization. FSHR-(533-555) was without effect on basal levels of cAMP and estradiol biosynthesis, indicating absence of toxicity at the concentrations tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat

Previous studies indicated that there is a separate hypothalamic control of follicle-stimulating hormone (FSH) release distinct from that of luteinizing hormone (LH). An FSH-releasing factor (FSHRF) was purified from rat and sheep hypothalami, but has not been isolated. We hypothesized that FSHRF might be an analogue of mammalian luteinizing hormone-releasing hormone (m-LHRH) and evaluated the activity of many analogues of m-LHRH and of the known LHRHs found in lower forms. Here we demonstrate that lamprey (l) LHRH-III has a potent, dose-related FSH- but not LH-releasing action on incubated hemipituitaries of male rats. l-LHRH-I on the other hand, had little activity to release either FSH or LH. m-LHRH was equipotent to l-LHRH-III to release FSH, but also had a high potency to release LH in contrast to l-LHRH-III that selectively released FSH. Chicken LHRH-II had considerable potency to release both LH and FSH, but no selectivity in its action. Salmon LHRH had much less potency than the others tested, except for l-LHRH-I, and no selectivity in its action. Because ovariectomized, estrogen, progesterone-treated rats are a sensitive in vivo assay for FSH- and LH-releasing activity, we evaluated l-LHRH-III in this assay and found that it had a completely selective stimulatory effect on FSH release at the two doses tested (10 and 100 pmols). Therefore, l-LHRH-III is a highly potent and specific FSH-releasing peptide that may enhance fertility in animals and humans. It may be the long sought after m-FSHRF.     

Chemokine receptor-ligand interactions measured using time-resolved fluorescence

Two G protein-coupled receptor subtypes (CXCR1 and CXCR2) mediate Interleukin-8 (IL8) action in cells. A nonradioactive lanthanide-chelate derivatized IL8 ligand was developed to measure the binding activity of the chemokine receptors, CXCR1 and CXCR2. Site-specific mutagenesis of the carboxyl-terminal serine of IL8 to cysteine resulted in a mutant IL8 (IL8-S72C) having a single free sulfhydryl. Using an iodoacetamide derivative of the Eu3+-chelate of N-(p-benzoic acid)diethylenetriamine-N,N',N"-tetraacetic acid (DTTA), incorporation of one Eu3+ per IL8 molecule ([Eu3+]IL8-S72C) was achieved. The dissociation constant for this conjugate was similar to that measured for [125I]IL8 ( approximately 2 nM) when measured by time-resolved fluorometry using CHO cell lines stably expressing CXCR1 or CXCR2 receptors. The sensitivity, stability, and high specific activity of europium-labeled IL8 demonstrate the usefulness of lanthanide-labeled proteins in the measurement of receptor-ligand interactions and may be extended to other peptide ligands.     

Hypothalamus-pituitary-adrenal activity during human sleep: a coordinating role for the limbic hippocampal system

It is well known that deglycosylation of gonadotropins by enzymatic or chemical procedures or by deletion of sites for N-linked glycosylation produces antagonistic analogs which are able to interact strongly with the receptor and to inhibit binding of the wild-type hormone. In the present study, we analyzed the antagonistic properties of a naturally occurring basic follicle-stimulating hormone (FSH) charge isoform obtained after high-resolution chromatofocusing of human anterior pituitary glycoprotein extracts. Coincubation of increasing amounts of this isoform with a highly purified human pituitary FSH preparation or with recombinant human FSH at doses equivalent to their corresponding ED50 for estradiol and tissue-type plasminogen activator (tPA) production, inhibited FSH-induced estrogen production and tPA enzyme activity by cultured rat granulosa cells in a dose-dependent manner. These inhibitory effects were apparently exerted at steps following 3',5'-cyclic adenosine monophosphate (cAMP) formation and did not involve activation of the protein kinase C pathway since: (a) at low doses, this basic FSH isoform moderately increased FSH-induced cAMP production by cultured rat granulosa cells; (b) coincubation of the antagonist isoform with dibutyryl cAMP completely inhibited the effects of this cAMP analog on estrogen and tPA production; (c) the isoform was able to stimulate production of cAMP in a human fetal cell line expressing the recombinant human FSH receptor, and (d) the inhibitory effects of the isoform were not affected by staurosporine, a protein kinase C inhibitor. The effects of this isoform upon dibutyryl cAMP-induced estrogen and tPA production were blocked by the addition of a highly specific antibody directed against human FSH, further demonstrating that the antagonistic effects observed were due to FSH-like molecules. In contrast to the inhibitory effects exhibited by this basic FSH isoform, a more acidic FSH charge variant consistently acted as an agonist of pituitary and recombinant FSH on both estrogen production and induction of tPA enzyme activity. These results indicate that the anterior pituitary gland normally produces FSH isoforms which act as either agonists or antagonists of FSH at the target cell level.     

Intracellular retention of mutant gonadotropin receptors results in loss of hormone binding activity of the follitropin receptor but not of the lutropin/choriogonadotropin receptor

It has recently been reported that Asp 397 of the rat lutropin/ choriogonadotropin receptor (rLHR) may be involved in transducing the signal from hormone binding to the stimulation of cAMP production. We examined the analogous region in the rat follitropin receptor (rFSHR) by substituting the Asp at position 404 (D404) of the rFSHR with either Glu (D404E), Ala (D404A), or Lys (D404K). Both in intact 293 cells and in detergent-solubilized extracts of 293 cells transiently transfected with the rFSHR constructs, only the wild type rFSHR exhibited detectable binding activity. Although the D404-substituted rFSHR mutants were visible on Western blots, in contrast to the wild type rFSHR which is present on Western blots as both mature and immature forms, only a single band comigrating with immature receptor was observed for the mutants. Furthermore, these mutants were sensitive to endoglycosidase H (Endo H), thus indicating that the mutant receptor proteins were retained intracellularly in the endoplasmic reticulum. To test whether the lack of binding of the D404-substituted rFSHR mutants was due to a perturbation of a binding site or to the intracellular retention of the mutants, a truncated rFSHR(t637) mutant, containing a cytoplasmic truncation that should not directly affect FSH binding, was examined. As with the D404-substitution mutants, rFSHR(t637) was stably expressed but sensitive to Endo H. Significantly, detergent-soluble extracts of cells expressing rFSHR(t637) were unable to bind FSH. From these results, we conclude that substitution of D404 of the rFSHR prevents hormone binding as a result of the intracellular retention of the mutants in the endoplasmic reticulum presumably in an incompletely folded state, as opposed to disruption of a hormone-binding site at D404. Comparable rLHR substitution (D397K) and truncation (t616) mutants were constructed and used to transfect 293 cells. For both rLHR(D397K) and rLHR(t616), human CG (hCG) binding to intact cells was not detectable, but high affinity hCG binding was observed in detergent-soluble extracts of the cells. Therefore, the rLHR differs from the rFSHR in that mutants of the rLHR that are retained in the endoplasmic reticulum have already been folded correctly and can bind hCG with high affinity as long as a hormone-binding site has not been perturbed by the mutation. In contrast, mutants of the rFSHR that are retained in the endoplasmic reticulum have not yet folded into a conformation that can bind hormone. This suggests a difference in the temporal pattern of folding between the two structurally related gonadotropin receptors. Our studies also demonstrate how mutagenesis studies of the rFSHR must be interpreted with caution, as FSHR mutants that are expressed but are retained intracellularly will most likely not be able to bind FSH even when a hormone-binding site has not been altered.     

Identification of residues in the neuronal alpha7 acetylcholine receptor that confer selectivity for conotoxin ImI

The neuronal-specific toxin alpha-conotoxin ImI (CTx ImI) has the sequence Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2, in which each cysteine forms a disulfide bridge to produce a constrained two-loop structure. To investigate the structural basis for bioactivity we mutated individual residues in CTx ImI and determined bioactivity. Bioactivity of the toxins was determined by their competition against 125I-labeled alpha-bungarotoxin binding to homomeric receptors containing alpha7 sequence in the major extracellular domain and 5HT-3 sequence elsewhere. The results reveal two regions in CTx ImI essential for binding to the alpha7/5HT-3 receptor. The first is the triad Asp-Pro-Arg in the first loop, where conservative mutations of each residue diminish affinity by 2-3 orders of magnitude. The second region is the lone Trp in the second loop, where an aromatic side chain is required. The overall results suggest that within the triad of the first loop, Pro positions the flanking Asp and Arg for optimal interaction with one portion of the binding site, while within the second loop, Trp stabilizes the complex through its aromatic ring.