Hormone interactions to Leu-rich repeats in the gonadotropin receptors. II. Analysis of Leu-rich repeat 4 of human luteinizing hormone/chorionic gonadotropin receptor

The luteinizing hormone receptor (LHR) consists of an approximately 350-amino acid-long N-terminal extracellular exodomain and a membrane-associated endodomain of similar size. Human chorionic gonadotropin (hCG) binds to the exodomain, and then hCG/exodomain complex is thought to make a secondary contact with the endodomain and generate hormone signals. The sequence alignment of the exodomain shows imperfectly matching eight to nine Leu-rich repeats (LRRs). In the preceding article (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435), we have shown that LRR2 and LRR4 are crucial for hormone binding. In this work, we have examined the residues of LRR4, in particular Leu(103) and Ile(105) in the putative beta strand. Our data show that Leu(103) and Ile(105) are involved in the specific, hydrophobic interaction of the LRR4 loop, likely to form the hydrophobic core. This loop is crucial for the structural integrity of all of the LRRs. In contrast, the downstream sequence consisting of Asn(107), Thr(108), Gly(109), and Ile(110) of LRR4 is crucial for cAMP induction but not for hormone binding, folding, and surface expression. This implicates, for the first time, its involvement in the interaction with the endodomain and signal generation. The evidence for the interaction is presented in the following article.     

Hormone interactions to Leu-rich repeats in the gonadotropin receptors. III. Photoaffinity labeling of human chorionic gonadotropin with receptor Leu-rich repeat 4 peptide

Human chorionic gonadotropin (hCG) binds to the extracellular N-terminal domain, exodomain, of its receptor, and the resulting hCG-exodomain complex is thought to modulate the membrane associated domain, endodomain, of the receptor to generate hormone signal. The bulk of the exodomain is speculated to assume a crescent structure consisting of eight to nine Leu-rich repeats (LRRs), which may provide the hormone contact sites. Unfortunately, little experimental evidence is available for the precise hormone contact points in the exodomain and the endodomain. The two preceding articles (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435; Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3436-3442) show that putative LRR2 and LRR4 are crucial for hormone binding. In particular, the N-terminal region of LRR4 assumes the hydrophobic core of the LRR4 loop, whereas the C-terminal region is crucial for signal generation. However, it is unclear whether LRR4 interacts hCG and the endodomain and how it might be involved in signal generation. In this article, our affinity labeling results present the first evidence that the N-terminal region of LRR4 interacts with hCG, preferentially the hCGalpha subunit and that the hCG/LRR4 complex interacts with exoloop 2 of the endodomain. This interaction offers a mechanism to generate hormone signal.     

Follicle-stimulating hormone interacts with exoloop 3 of the receptor

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of approximately 330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of (580)KVPLITVSKAK(590) was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH alpha but not FSH beta. This is in contrast to photoaffinity labeling of FSH beta by the peptide mimic of the N-terminal region of the receptor. Leu(583) and Ile(584) are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu(583) and Ile(584) side chains appear to project in opposite directions. Ile(584) appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu(583) is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu(583) improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile(584) and Lys(590) abolished the cAMP and IP induction. Our results open a logical question whether Leu(583), Ile(584), and Lys(590) interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation.     

The differential binding affinities of the luteinizing hormone (LH)/choriogonadotropin receptor for LH and choriogonadotropin are dictated by different extracellular domain residues

The high degree of amino acid sequence homology and the divergent ligand binding affinities of the rat (r) and human (h) LH receptors (LHRs) allowed us to identify amino acid residues of their extracellular domain that are responsible for the different binding affinities of bovine (b) and hLH, and human choriogonadotropin (hCG) to the hLHR and rLHR. Because of the proposed importance of the beta-sheets of the leucine-rich repeats (LRRs) of the extracellular domain of the LHR on hormone binding, we examined 10 divergent residues present in these regions by analyzing two complementary sets of mutants in which hLHR residues were substituted with the corresponding rLHR residues and vice versa. These experiments resulted in the identification of a single residue (a Ile or Ser in the C-terminal end of LRR2 of the hLHR or rLHR, respectively) that is important for hLH binding affinity. Surprisingly, however, this residue does not affect hCG or for bLH binding affinity. In fact, the results obtained with bLH and hCG show that several of the divergent residues in the beta-sheets of LRR1-9 affect bLH binding affinity, but none of them affect hCG binding affinity. Importantly, our results also emphasize the involvement of residues outside of the beta-sheets of the LRRs of the LHR in ligand binding affinity. This finding has to be considered in future models of the interaction of LH/CG with the LHR.     

Trans-activation of mutant follicle-stimulating hormone receptors selectively generates only one of two hormone signals

Previously, we reported that a liganded LH receptor (LHR) is capable of activating itself (cis-activation) and other nonliganded LHRs to induce cAMP (trans-activation). Trans-activation of the LHR raises two crucial questions. Is trans-activation unique to LHR or common to other G protein-coupled receptors? Does trans-activation stimulate phospholipase Cbeta as it does adenylyl cyclase? To address these questions, two types of novel FSH receptors (FSHRs) were constructed, one defective in hormone binding and the other defective in signal generation. The FSHR, a G protein-coupled receptor, comprises two major domains, the N-terminal extracellular exodomain that binds the hormone and the membrane-associated endodomain that generates the hormone signals. For signal defective receptors, the exodomain was attached to glycosyl phosphatidylinositol (ExoGPI) or the transmembrane domain of CD8 immune receptor (ExoCD). ExoGPI and ExoCD can trans-activate another nonliganded FSH. Surprisingly, the trans-activation generates a signal to activate either adenylyl cyclase or phospholipase Cbeta, but not both. These results indicate that trans-activation in these mutant receptors is selective and limited in signal generation, thus providing new approaches to investigating the generation of different hormone signals and a novel means to selectively generate a particular hormone signal. Our data also suggest that the FSHR's exodomain could not trans-activate LHR.     

The role of the hinge region of the luteinizing hormone receptor in hormone interaction and signal generation

Luteinizing hormone receptor, a G protein-coupled receptor, consists of two halves, the N-terminal extracellular hormone binding domain (exodomain) and the C-terminal membrane-associated, signal-generating domain (endodomain). The exodomain has seven to nine Leu-rich repeats, which are generally thought to form a 1/3 donut-like structure and interact with human choriogonadotropin (hCG). The resulting hCG-exodomain complex adjusts the structure and its association with the endodomain, which results in signal generation in the endodomain. It is unclear whether the rigid 1/3 donut structure could provide the agility and versatility of this dynamic action. In addition, there is no clue as to where the endodomain contact point (the signal modulator) in the exodomain is. To address these issues, the exodomain was examined by Ala scan and multiple substitutions, while receptor peptides were used for photoaffinity labeling and affinity cross-linking. Our results show that the C-flanking sequence (hinge region), Thr(250)-Gln(268), of the Leu-rich repeats (LRRs) specifically interacts with hCG, preferentially hCGalpha. This interaction is inhibited by exoloop 2 of the endodomain but not by exoloops 1 and 3, suggesting an intimate relationship between Thr(250)-Gln(268), exoloop 2, and hCG. Taken together, our observations in this article suggest a new paradigm that the LRRs contact the front of hCG, while both flanking regions of the LRRs interact with the sides of hCG. This would trap hCG in the 1/3 donut structure of the LRRs and enhance the binding affinity. In addition, mutations of conserved Ser(255) in the sequence can constitutively activate the receptor. This provides a clue for the signal modulator in the exodomain. In contrast, a phenyl or phenolic group is necessary at conserved Tyr(253) for targeting the receptor to the surface.     

Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity

Glycoprotein hormone receptors [thyrotropin (TSHr), luteinizing hormone/chorionic gonadotropin (LH/CGr), follicle stimulating hormone (FSHr)] are rhodopsin-like G protein-coupled receptors with a large extracellular N-terminal portion responsible for hormone recognition and binding. In structural models, this ectodomain is composed of two cysteine clusters flanking nine leucine-rich repeats (LRRs). The LRRs form a succession of beta-strands and alpha-helices organized into a horseshoe-shaped structure. It has been proposed that glycoprotein hormones interact with residues of the beta-strands making the concave surface of the horseshoe. Gain-of-function homology scanning of the beta-strands of glycoprotein hormone receptors allowed identification of the critical residues responsible for the specificity towards human chorionic gonadotropin (hCG). Substitution of eight or two residues of the LH/CGr into the TSHr or FSHr, respectively, resulted in constructs displaying almost the same affinity and sensitivity for hCG as wild-type LH/CGr. Molecular dynamics simulations and additional site-directed mutagenesis provided a structural rationale for the evolution of binding specificity in this duplicated gene family.     

Super-motifs and evolution of tandem leucine-rich repeats within the small proteoglycans--biglycan, decorin, lumican, fibromodulin, PRELP, keratocan, osteoadherin, epiphycan, and osteoglycin

Leucine-rich repeats (LRRs) with 20-30 amino acids in unit length are present in many proteins from prokaryotes to eukaryotes. The LRR-containing proteins include a family of nine small proteoglycans, forming three distinct subfamilies: class I contains biglycan/PG-I and decorin/PG-II; class II: lumican, fibromodulin, PRELP, keratocan, and osteoadherin; and class III: epiphycan/PG-Lb and osteoglycin or osteoinductive factor. Comparative sequence analysis of the 34 available protein sequences reveals that these proteoglycans have two types of LRRs, which we call S and T. The type S LRR is 21 residues long and has the consensus sequence of xxaPzxLPxxLxxLxLxxNxI. The type T LRR has 26 residues; its consensus sequence is zzxxaxxxxFxxaxxLxxLxLxxNxL. In both "x" indicates variable residue; "z" is frequently a gap; "a" is Val, Leu, or Ile; and I is Ile or Leu. These type S and TLRRs are ordered into two super-motifs--STT with about 73 residues in classes I and II and ST with about 47 residues in class III. The 12 LRRs in the small proteoglycans of I and II are best represented as (STT)4; the seven LRRs of class III as (ST)T(ST)2. Our analyses indicate that classes I/II and III evolved along different paths after the establishment of the precursor ST, and classes I and II also diverged after the establishment of the precursor (STT)4.     

Ligand selectivity of gonadotropin receptors. Role of the beta-strands of extracellular leucine-rich repeats 3 and 6 of the human luteinizing hormone receptor

The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hFSH-R) and human luteinizing hormone/chorionic gonadotropin receptor (hLH-R) is determined by their approximately 350 amino acid-long N-terminal receptor exodomains that allow the mutually exclusive binding of human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) when these hormones are present in physiological concentrations. The exodomains of each of these receptors consist of a nine-leucine-rich repeat-containing subdomain (LRR subdomain) flanked by N- and C-terminal cysteine-rich subdomains. Chimeric receptors, in which the structural subdomains of the hFSH-R exodomain were substituted with those of the hLH-R, showed a similar high responsiveness to human chorionic gonadotropin (hCG) and hLH as long as they harbored the LRR subdomain of the hLH-R. In addition, these chimeric receptors showed no responsiveness to hFSH. The LRR subdomains of the gonadotropin receptor exodomains are predicted to adopt a horseshoe-like conformation, of which the hormone-binding concave surface is composed of nine parallel beta-strands. Receptors in which individual beta-strands of the hFSH-R were replaced with the corresponding hLH-R sequences revealed that hCG and hLH selectivity is predominantly determined by hLH-R beta-strands 3 and 6. A mutant receptor in which the hFSH-R beta-strands 3 and 6 were substituted simultaneously with their hLH-R counterparts displayed a responsiveness to hCG and hLH similar to that of the wild type hLH-R. Responsiveness to hFSH was not affected by most beta-strand substitutions, suggesting the involvement of multiple low-impact determinants for this hormone.     

Comparative structural analysis of the binding domain of follicle stimulating hormone receptor

Proteins with leucine-rich repeats (LRRs) specialize in mediating protein-protein interactions. The hormone binding portion of the receptor for follicle stimulating hormone (FSH) is an LRR protein by sequence, and the crystal structure of this domain from human FSH receptor in a complex with FSH shows that it does indeed have an LRR structure. It differs from other LRR domains, however, in being an all-beta protein composed of highly irregular repeats and having only slight overall curvature. Despite these distinctions and a superficial resemblance to beta-helical proteins, the binding domain of FSH receptor clearly is an LRR protein. The structure does consist of two parts with distinctively different curvatures. Comparison with the structures of other LRR-containing proteins shows a correlation between curvature and main-chain hydrogen bonding pattern of the parallel beta-sheet. The hormone-binding site is located at the concave surface of the receptor structure, a feature common to proteins with LRR motifs. Analysis of the ligand-binding site of LRR-containing proteins reveals that they generally utilize extensive interface area and a large number of charged residues to facilitate high-affinity protein-protein interactions.     

Orientation of follicle-stimulating hormone (FSH) subunits complexed with the FSH receptor. Beta subunit toward the N terminus of exodomain and alpha subunit to exoloop 3

Follicle-stimulating hormone (FSH) comprises an alpha subunit and a beta subunit, whereas the FSH receptor consists of two halves with distinct functions: the N-terminal extracellular exodomain and C-terminal membrane-associated endodomain. FSH initially binds to exodomain, and the resulting FSH/exodomain complex modulates the endodomain and generates signal. However, it has been difficult to determine which subunit of FSH contacts the exodomain or endodomain and in what orientation FSH interacts with them. To address these crucial issues, the receptor was Ala-scanned and the hormone subunits were probed with photoaffinity labeling with receptor peptides corresponding to the N-terminal region of the exodomain and exoloop 3 of the endodomain. Our results show that both regions of the receptors are important for hormone binding and signal generation. In addition, the FSH beta subunit is specifically labeled with the N-terminal peptide, whereas the alpha subunit is labeled with the exoloop 3 peptide. These contrasting results show that the FSH beta subunit is close to the N-terminal region and that the alpha subunit is projected toward exoloop 3 in the endodomain. The results raise the fundamental question whether the alpha subunit, common among the glycoprotein hormones, plays a major role in generating the hormone signal common to all glycoprotein hormones.     

Expression of messenger RNA for gonadotropin receptor in the granulosa layer during the ovulatory cycle of hens.

The present experiments were conducted to evaluate the mRNA levels of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) in granulosa layers during the ovulatory cycle of hens, in relation to the release of LH and steroid hormones. After the release of LH, progesterone (P4) and estradiol-17beta (E2), found 4-5 h before ovulation, LHR and FSHR mRNA levels were observed to decrease in the granulosa layers of the largest (F1) and second largest (F2) preovulatory follicles, with the greatest in the LHR mRNA level of F1. P4 concentrations in the granulosa layers of F1 and F2 increased 4-5 h before ovulation, with greater in F1 than in F2. F2 concentrations in the theca layers were greater in F2 than in F1 throughout the ovulatory cycle. Also, the injection of ovine LH caused decreases in the mRNA levels of LHR and FSHR in the granulosa layers. However, these decreases were abolished by the injection of aminoglutethimide, an inhibitor of steroid synthesis. These results suggest that in hen granulosa cells, the mRNA levels of not only LHR but also FSHR are down-regulated by LH and the down-regulation may be mediated steroid hormones.     

Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in β-strands 2 (Lys⁷⁴), 4 (Tyr¹²⁴, Asn¹²⁹, and Thr¹³⁰), and 5 (Asp¹⁵⁰ and Asp¹⁵³) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in β-strands 2 (Ser⁷⁸) and 8 (Asp²²⁴ and Ser²²⁶) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.     

Expression of luteinizing hormone and follicle-stimulating hormone receptor in the dog prostate

A possible role for gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the prostate physiology has been suggested in humans and rats. This study aimed at investigating the presence of receptors for LH and FSH (LHR and FSHR) in the canine prostate. Prostates were collected at post mortem from 6 clinically healthy, sexually intact beagles free from any prostatic disorder. Tissue was sampled from dorsal, middle and ventral regions of each prostate. Immunohistochemical localization was performed on wax-embedded sections using polyclonal antibodies for LHR or FSHR. The pattern and intensity of staining in the parenchyma (glandular epithelium) and stroma were determined using a semiquantitative histologic assessment. Receptors for LH and FSH were consistently present in both the glandular epithelium and the stroma in all tissue samples examined. Expression for both receptors was higher in the glandular epithelium than the stroma of all prostatic regions (P < 0.001). In the glandular epithelium, LHR (P < 0.01) and FSHR (P < 0.05) expression was lower in the lateral than the other regions, and there was no difference between dorsal and ventral regions. However, variations in the expression for LHR and FSHR among prostatic regions were not found in the stroma. These findings have demonstrated that LHR and FSHR are expressed in the dog prostate, and the variation observed in their levels of expression among its regions and tissue layers suggests a potential role of gonadotrophins LH and FSH in the regulation of the prostate physiology, particularly the glandular epithelium.     

Piscine glycoprotein hormone (gonadotropin and thyrotropin) receptors: a review of recent developments

Similar to the higher vertebrates, the pituitary in bony fishes express three glycoprotein hormones: thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In addition to the appropriate secretion of these hormones, the timely and quantitative expression of their specific receptors (TSHR, FSHR and LHR) in the target tissues is an essential requirement for their physiological action. In fishes that constitute a very diverse group of vertebrates, there are only a few published reports of primary structure of these receptors although other examples have been communicated briefly. This review will summarize these reports as well as to describe the insights gained from what is known about the mammalian receptors. The structural organization of the fish receptors (as deduced from the encoding cDNAs) is highly homologous to the higher vertebrate receptors in that there is a 7-pass transmembrane region and an N-terminal extracellular domain, which contributes to ligand specificity. In mammals, the FSHR and the TSHR genes are composed of 10 exons whereas the LHR gene is composed of 11 exons. The position of the 'extra intron' is conserved in the catfish LHR gene. In the mammals, the transmembrane domain of each of the three glycoprotein hormone receptors is encoded by a single exon, however, in the salmon genes and homologous invertebrate genes, this portion of the receptor is encoded by multiple exons. In general, the tissue-specific expression of these receptors is similar to that seen in mammals, however, the gonadal expression of TSHR in the striped bass and sunrise sculpin and the renal expression of LHR in the channel catfish are unique.     

A nested leucine rich repeat (LRR) domain: The precursor of LRRs is a ten or eleven residue motif

Background  

Leucine rich repeats (LRRs) are present in over 60,000 proteins that have been identified in viruses, bacteria, archae, and eukaryotes. All known structures of repeated LRRs adopt an arc shape. Most LRRs are 20-30 residues long. All LRRs contain LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. Seven classes of LRRs have been identified. However, other LRR classes remains to be characterized. The evolution of LRRs is not well understood.     

Studies with chimeras of the gonadotropin receptors reveal the importance of third intracellular loop threonines on the formation of the receptor/nonvisual arrestin complex

Using chimeras and more discrete exchange mutations of the rat (r) and human (h) gonadotropin receptors, we had previously identified multiple noncontiguous residues of the lutropin (LHR) and follitropin (FSHR) receptors that dictate their rates of internalization. Since the internalization of the LHR and the FSHR is driven by their abilities to associate with the nonvisual arrestins, we hypothesized that one or more of the residues previously identified by the internalization assays are involved in the formation of the receptor/nonvisual arrestin complex. In the studies reported herein, we tested this hypothesis by measuring the association of arrestin-3 with a large number of rLHR/hLHR and rFSHR/hFSHR exchange mutants that affect internalization. The results presented show that the same residues that dictate the rate of internalization of these two receptor pairs affect their ability to associate with arrestin-3. Although these residues are located in distinct topological domains, our analyses show that threonine residues in the third intracellular loop of both receptor pairs are particularly important for the formation of the receptor/arrestin-3 complexes and internalization. We conclude that the different rates of internalization of the gonadotropin receptors are dictated by their different abilities to associate with the nonvisual arrestins and that this association is, in turn, largely dictated by the presence of threonine residues in their third intracellular loops.     

Transcriptional profiling of Actinobacillus pleuropneumoniae under iron-restricted conditions

Background

Toll-like receptors (TLRs) play a central role in innate immunity. TLRs are membrane glycoproteins and contain leucine rich repeat (LRR) motif in the ectodomain. TLRs recognize and respond to molecules such as lipopolysaccharide, peptidoglycan, flagellin, and RNA from bacteria or viruses. The LRR domains in TLRs have been inferred to be responsible for molecular recognition. All LRRs include the highly conserved segment, LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. There are seven classes of LRRs including "typical" ("T") and "bacterial" ("S"). All known domain structures adopt an arc or horseshoe shape. Vertebrate TLRs form six major families. The repeat numbers of LRRs and their "phasing" in TLRs differ with isoforms and species; they are aligned differently in various databases. We identified and aligned LRRs in TLRs by a new method described here.

Results

The new method utilizes known LRR structures to recognize and align new LRR motifs in TLRs and incorporates multiple sequence alignments and secondary structure predictions. TLRs from thirty-four vertebrate were analyzed. The repeat numbers of the LRRs ranges from 16 to 28. The LRRs found in TLRs frequently consists of LxxLxLxxNxLxxLxxxxF/LxxLxx ("T") and sometimes short motifs including LxxLxLxxNxLxxLPx(x)LPxx ("S"). The TLR7 family (TLR7, TLR8, and TLR9) contain 27 LRRs. The LRRs at the N-terminal part have a super-motif of STT with about 80 residues. The super-repeat is represented by STTSTTSTT or _TTSTTSTT. The LRRs in TLRs form one or two horseshoe domains and are mostly flanked by two cysteine clusters including two or four cysteine residue.

Conclusion

Each of the six major TLR families is characterized by their constituent LRR motifs, their repeat numbers, and their patterns of cysteine clusters. The central parts of the TLR1 and TLR7 families and of TLR4 have more irregular or longer LRR motifs. These central parts are inferred to play a key role in the structure and/or function of their TLRs. Furthermore, the super-repeat in the TLR7 family suggests strongly that "bacterial" and "typical" LRRs evolved from a common precursor.     

Discovery of substituted benzamides as follicle stimulating hormone receptor allosteric modulators

Follicle-stimulating hormone (FSH), acting on its receptor (FSHR), plays a pivotal role in the stimulation of follicular development and maturation. Multiple injections of protein formulations are used during clinical protocols for ovulation induction and for in vitro fertilization that are followed by a selection of assisted reproductive technologies. In order to increase patient convenience and compliance several research groups have searched for orally bioavailable FSH mimetics for innovative fertility medicines. We report here the discovery of a series of substituted benzamides as positive allosteric modulators (PAM) targeting FSHR. Optimization of this series has led to enhanced activity in primary rat granulosa cells, as well as remarkable selectivity against the closely related luteinizing hormone receptor (LHR) and thyroid stimulating hormone receptor (TSHR). Two modulators, 9j and 9k, showed promising in vitro and pharmacokinetic profiles.