Data set for transcriptional response to depletion of the Shoc2 scaffolding protein.

The Suppressor of Clear, Caenorhabditis elegans Homolog (SHOC2) is a scaffold protein that positively modulates activity of the RAS/ERK1/2 MAP kinase signaling cascade. We set out to understand the ERK1/2 pathway transcriptional response transduced through the SHOC2 scaffolding module. This data article describes raw gene expression within triplicates of kidney fibroblast-like Cos1 cell line expressing non-targeting shRNA (Cos-NT) and triplicates of Cos1 cells depleted of SHOC2 using shRNA (Cos-LV1) upon activation of ERK1/2 pathway by the Epidermal Growth Factor Receptor (EGFR). The data referred here is available in NCBI׳s Gene Expression Omnibus (GEO), accession GEO: GSE67063 as well as NCBI׳s Sequence Read Archive (SRA), accession SRA: SRP056324. A complete analysis of the results can be found in "Shoc2-tranduced ERK1/2 motility signals - Novel insights from functional genomics"(Jeoung et al., 2016) [1].

Shoc2-tranduced ERK1/2 motility signals--Novel insights from functional genomics.

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway plays a central role in defining various cellular fates. Scaffold proteins modulating ERK1/2 activity control growth factor signals transduced by the pathway. Here, we analyzed signals transduced by Shoc2, a critical positive modulator of ERK1/2 activity. We found that loss of Shoc2 results in impaired cell motility and delays cell attachment. As ERKs control cellular fates by stimulating transcriptional response, we hypothesized that the mechanisms underlying changes in cell adhesion could be revealed by assessing the changes in transcription of Shoc2-depleted cells. Using quantitative RNA-seq analysis, we identified 853 differentially expressed transcripts. Characterization of the differentially expressed genes showed that Shoc2 regulates the pathway at several levels, including expression of genes controlling cell motility, adhesion, crosstalk with the transforming growth factor beta (TGFß) pathway, and expression of transcription factors. To understand the mechanisms underlying delayed attachment of cells depleted of Shoc2, changes in expression of the protein of extracellular matrix (lectin galactoside-binding soluble 3-binding protein; LGALS3BP) were functionally analyzed. We demonstrated that delayed adhesion of the Shoc2-depleted cells is a result of attenuated expression and secretion of LGALS3BP. Together our results suggest that Shoc2 regulates cell motility by modulating ERK1/2 signals to cell adhesion.

Estrogen Receptor Alpha (ESR1)-Dependent Regulation of the Mouse Oviductal Transcriptome.

Estrogen receptor-α (ESR1) is an important transcriptional regulator in the mammalian oviduct, however ESR1-dependent regulation of the transcriptome of this organ is not well defined, especially at the genomic level. The objective of this study was therefore to investigate estradiol- and ESR1-dependent regulation of the transcriptome of the oviduct using transgenic mice, both with (ESR1KO) and without (wild-type, WT) a global deletion of ESR1. Oviducts were collected from ESR1KO and WT littermates at 23 days of age, or ESR1KO and WT mice were treated with 5 IU PMSG to stimulate follicular development and the production of ovarian estradiol, and the oviducts collected 48 h later. RNA extracted from whole oviducts was hybridized to Affymetrix Genechip Mouse Genome 430-2.0 arrays (n = 3 arrays per genotype and treatment) or reverse transcribed to cDNA for analysis of the expression of selected mRNAs by real-time PCR. Following microarray analysis, a statistical two-way ANOVA and pairwise comparison (LSD test) revealed 2428 differentially expressed transcripts (DEG's, P < 0.01). Genotype affected the expression of 2215 genes, treatment (PMSG) affected the expression of 465 genes, and genotype x treatment affected the expression of 438 genes. With the goal of determining estradiol/ESR1-regulated function, gene ontology (GO) and bioinformatic pathway analyses were performed on DEG's in the oviducts of PMSG-treated ESR1KO versus PMSG-treated WT mice. Significantly enriched GO molecular function categories included binding and catalytic activity. Significantly enriched GO cellular component categories indicated the extracellular region. Significantly enriched GO biological process categories involved a single organism, modulation of a measurable attribute and developmental processes. Bioinformatic analysis revealed ESR1-regulation of the immune response within the oviduct as the primary canonical pathway. In summary, a transcriptomal profile of estradiol- and ESR1-regulated gene expression and related bioinformatic analysis is presented to increase our understanding of how estradiol/ESR1 affects function of the oviduct, and to identify genes that may be proven as important regulators of fertility in the future.

Spatial control of Shoc2-scaffold-mediated ERK1/2 signaling requires remodeling activity of the ATPase PSMC5.

The scaffold protein Shoc2 accelerates activity of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) pathway. Mutations in Shoc2 result in Noonan-like RASopathy, a developmental disorder with a wide spectrum of symptoms. The amplitude of the ERK1/2 signals transduced through the complex is fine-tuned by the HUWE1-mediated ubiquitylation of Shoc2 and its signaling partner RAF-1. Here, we provide a mechanistic basis of how ubiquitylation of Shoc2 and RAF-1 is controlled. We demonstrate that the newly identified binding partner of Shoc2, the (AAA+) ATPase PSMC5, triggers translocation of Shoc2 to endosomes. At the endosomes, PSMC5 displaces the E3 ligase HUWE1 from the scaffolding complex to attenuate ubiquitylation of Shoc2 and RAF-1. We show that a RASopathy mutation that changes the subcellular distribution of Shoc2 leads to alterations in Shoc2 ubiquitylation due to the loss of accessibility to PSMC5. In summary, our results demonstrate that PSMC5 is a new and important player involved in regulating ERK1/2 signal transmission through the remodeling of Shoc2 scaffold complex in a spatially-defined manner.

A Novel SHOC2 Variant in Rasopathy.

Rasopathies are a group of genetic disorders caused by germline mutations in multiple genes of the Extracellular signal-Regulated Kinases 1 and 2 (ERK1/2) pathway. The only previously identified missense mutation in SHOC2, a scaffold protein of the ERK1/2 pathway, led to Noonan-like syndrome with loose anagen hair. Here, we report a novel mutation in SHOC2(c.519G>A; p.M173I) that leads to a Rasopathy with clinical features partially overlapping those occurring in Noonan and cardiofaciocutaneous syndromes. Studies to clarify the significance of this SHOC2 variant revealed that the mutant protein has impaired capacity to interact with protein phosphatase 1c (PP1c), leading to insufficient activation of RAF-1 kinase. This SHOC2 variant thus is unable to fully rescue ERK1/2 activity in cells depleted of endogenous SHOC2. We conclude that SHOC2 mutations can cause a spectrum of Rasopathy phenotypes in heterozygous individuals. Importantly, our work suggests that individuals with mild Rasopathy symptoms may be underdiagnosed.

HUWE1 is a molecular link controlling RAF-1 activity supported by the Shoc2 scaffold.

Scaffold proteins play a critical role in controlling the activity of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Shoc2 is a leucine-rich repeat scaffold protein that acts as a positive modulator of ERK1/2 signaling. However, the precise mechanism by which Shoc2 modulates the activity of the ERK1/2 pathway is unclear. Here we report the identification of the E3 ubiquitin ligase HUWE1 as a binding partner and regulator of Shoc2 function. HUWE1 mediates ubiquitination and, consequently, the levels of Shoc2. Additionally, we show that both Shoc2 and HUWE1 are necessary to control the levels and ubiquitination of the Shoc2 signaling partner, RAF-1. Depletion of HUWE1 abolishes RAF-1 ubiquitination, with corresponding changes in ERK1/2 pathway activity occurring. Our results indicate that the HUWE1-mediated ubiquitination of Shoc2 is the switch that regulates the transition from an active to an inactive state of the RAF-1 kinase. Taken together, our results demonstrate that HUWE1 is a novel player involved in regulating ERK1/2 signal transmission through the Shoc2 scaffold complex.

Visualizing of signaling proteins on endosomes utilizing knockdown and reconstitution approach.

Spatial distribution of intracellular signaling molecules and assembly of signaling complexes are yet to be fully understood. Studies of signaling events in time or space present a particular challenge due to the adverse effects that overexpression of signaling proteins may have on their functions and localization. To follow the distribution of signaling proteins in living cells we developed a methodology named knockdown and reconstitution (KDAR) that allows one to visualize proteins at levels of expression that are close to physiological. This methodology provides a stable expression of "endogenous" shRNA for long-term silencing of the targeted gene and simultaneous expression of a DNA cassette coding for a fluorescently labeled protein, which is insensitive to the targeting shRNA. In this chapter we discuss the needed reagents and outline two experimental approaches to generate KDAR stable cell lines. First, we demonstrate how the plasmid-mediated KDAR approach is successfully utilized to visualize spatial distribution of the GFP-labeled MEK2 in living cells. We then show how the lentivirus-mediated KDAR approach is used to reconstitute and visualize expression of the ERK1/2 scaffold protein Shoc2.

Functional Integration of the Conserved Domains of Shoc2 Scaffold.

Shoc2 is a positive regulator of signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Shoc2 is also proposed to interact with RAS and Raf-1 in order to accelerate ERK1/2 activity. To understand the mechanisms by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor receptor (EGFR), we dissected the role of Shoc2 structural domains in binding to its signaling partners and its role in regulating ERK1/2 activity. Shoc2 is comprised of two main domains: the 21 leucine rich repeats (LRRs) core and the N-terminal non-LRR domain. We demonstrated that the N-terminal domain mediates Shoc2 binding to both M-Ras and Raf-1, while the C-terminal part of Shoc2 contains a late endosomal targeting motif. We found that M-Ras binding to Shoc2 is independent of its GTPase activity. While overexpression of Shoc2 did not change kinetics of ERK1/2 activity, both the N-terminal and the LRR-core domain were able to rescue ERK1/2 activity in cells depleted of Shoc2, suggesting that these Shoc2 domains are involved in modulating ERK1/2 activity.

Hematopoetic prostaglandin D synthase: an ESR1-dependent oviductal epithelial cell synthase.

Oviductal disease is a primary cause of infertility, a problem that largely stems from excessive inflammation of this key reproductive organ. Our poor understanding of the mechanisms regulating oviductal inflammation restricts our ability to diagnose, treat, and/or prevent oviductal disease. Using mice, our objective was to determine the spatial localization, regulatory mechanism, and functional attributes of a hypothesized regulator of oviductal inflammation, the hematopoietic form of prostaglandin D synthase (HPGDS). Immunohistochemistry revealed specific localization of HPGDS to the oviduct's epithelium. In the isthmus, expression of HPGDS was consistent. In the ampulla, expression of HPGDS appeared dependent upon stage of the estrous cycle. HPGDS was expressed in the epithelium of immature and cycling mice but not in the oviducts of estrogen receptor α knockouts. Two receptor subtypes bind PGD2: PGD2 receptor and G protein-coupled receptor 44. Expression of mRNA for Ptgdr was higher in the epithelial cells (EPI) than in the stroma (P < 0.05), whereas mRNA for Gpr44 was higher in the stroma than epithelium (P < 0.05). Treatment of human oviductal EPI with HQL-79, an inhibitor of HPGDS, decreased cell viability (P < 0.05). Treatment of mice with HQL-79 increased mRNA for chemokine (C-C motif) ligands 3, 4, and 19; chemokine (C-X-C motif) ligands 11 and 12; IL-13 and IL-17B; and TNF receptor superfamily, member 1b (P < 0.02 for each mRNA). Overall, these results suggest that HPGDS may play a role in the regulation of inflammation and EPI health within the oviduct.

In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1, PGE2) prevent luteolysis in cows. II: mRNA for PGF2α, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4 prostanoid receptors in luteal tissue.

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every 4h inhibited luteolysis in ewes by altering luteal mRNA for luteinizing hormone (LH) receptors and unoccupied and occupied luteal LH receptors. However, estradiol-17ß or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in cows, but infusion of estradiol+PGE(2) inhibited luteolysis. In contrast, intra-luteal implants containing PGE(1) or PGE(2) in Angus or Brahman cows also inhibited the decline in circulating progesterone, mRNA for LH receptors, and loss of unoccupied and occupied receptors for LH to prevent luteolysis. The objective of this experiment was to determine how intra-luteal implants of PGE(1) or PGE(2) alter mRNA for prostanoid receptors and how this could influence luteolysis in Brahman or Angus cows. On day-13 Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Corpora lutea slices were analyzed for mRNA for prostanoid receptors (FP, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4) by RT-PCR. Day-13 Angus cow luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE(1) and PGE(2) up-regulated FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed. EP1 mRNA was not altered by any treatment. PGE(1) and PGE(2) down-regulated EP2 and EP4 mRNA compared to day-19 Vehicle controls regardless of breed. PGE(1) or PGE(2) up-regulated mRNA EP3B receptor subtype compared to day-19 Vehicle control cows regardless of breed. The similarities in relative gene expression profiles induced by PGE(1) and PGE(2) support their agonistic effects. We conclude that both PGE(1) and PGE(2) may prevent luteolysis by altering expression of mRNA for prostanoid receptors, which is correlated with changes in luteal mRNA for LH receptors reported previously in these same cows to prevent luteolysis.

Cyclic regulation of apoptotic gene expression in the mouse oviduct.

The oviduct is a dynamic structure whose function relies upon cyclic changes in the morphology of both ciliated and secretory luminal epithelial cells. Unfortunately, infection of these epithelial cells by sexually transmitted pathogens can lead to pelvic inflammatory disease, ectopic pregnancies and infertility. The disruption of normal, cyclic apoptosis in the oviducal epithelium appears to be a causal factor of oviducal pathology and therefore, these pathways represent a potential target for diagnosis and therapeutic intervention. The objective of this study was to determine the pattern of expression for apoptotic genes in the oviduct of the naturally cycling mouse, generating fundamental information that can be applied to the development of animal models for research and the identification of targets for disease intervention. Whole oviducts were collected from regular cycling mice killed at 1p.m. on each day of the oestrous cycle and the expression of 84 apoptotic genes determined by targeted PCR super-array. Intact and cleaved caspases were then evaluated by western blotting. The expression of mRNA for genes classified as pro-apoptotic (Bad, Bak1 and Bok) and anti-apoptotic (Bag3, Bnip2 and Xiap) was regulated by day (P < 0.05). Differences in the temporal expression of several p53-related genes (Trp53bp2, Trp53inp1 and Trp73), those specific to the TNF superfamily (Tnfrsf10 and Tnfsf10b) and one caspase (Casp14) were also observed (P < 0.05). The cleaved forms of Caspases-3, -6 and -12 were all detected throughout the oestrous cycle. These results represent the first pathway-wide analysis of apoptotic gene expression in the murine oviduct.

Methodology matters: IVF versus ICSI and embryonic gene expression.

The use of assisted reproduction treatment, especially intracytoplasmic sperm injection (ICSI), is now linked to a range of adverse consequences, the aetiology of which remains largely undefined. Our objective of this study was to determine differences in gene expression of blastocysts generated by ICSI as well as ICSI with artificial oocyte activation (ICSI-A) versus the less manipulative IVF, providing fundamental genetic information that can be used to aid in the diagnosis or treatment of those adversely affected by assisted reproduction treatment, as well as stimulate research to further refine these techniques. Murine blastocysts were generated by ICSI, ICSI-A and IVF, and processed for a microarray-based analysis of gene expression. Ten blastocysts were pooled for each procedure and three independent replicates generated. The data were then processed to determine differential gene expression and to identify biological pathways affected by the procedures. In blastocysts derived by ICSI versus IVF, the expression of 197 genes differed (P < 0.01). In blastocysts derived by ICSI-A versus IVF and ICSI-A versus ICSI, the expression of 132 and 65 genes differed respectively (P < 0.01). Procedural-induced changes in genes regulating specific biological pathways revealed some consistency to known adverse consequences. Detailed investigation of procedure-specific dysfunction is therefore warranted.

Production and binding of endothelin-2 (EDN2) in the rat ovary: endothelin receptor subtype A (EDNRA)-mediated contraction.

Endothelin-2 (EDN2)-mediated contraction has been proposed as a final mechanical signal facilitating ovulation. The objectives herein were to determine (1) whether ovarian endothelins were increased before ovulation; (2) whether a specific endothelin-converting enzyme (ECE) was mediating their production; (3) which receptor was facilitating ovarian contraction; and (4) whether receptor-specific antagonism affected ovulation. Follicular development was induced in immature rats with 10 IU pregnant mare serum gonadotrophin (PMSG) and the ovulatory cascade was initiated 48 h later with 10 IU human chorionic gonadotrophin (hCG). In Experiment 1, an immunoassay revealed that the ovarian concentration of endothelin peptide was increased 7-fold 12 h after hCG when compared with 48 h after PMSG (P < 0.05). In Experiment 2, real-time PCR indicated that mRNA for Ece1, but not Ece2, was increased in granulosa cells collected 12 h after hCG when compared with those collected before the ovulatory stimulus (P < 0.05). In Experiment 3, isometric tension analysis revealed that the contractile effect of EDN2 was mediated by endothelin receptor A (EDNRA), not B (EDNRB). In Experiment 4, no effect was observed on the rate of ovulation when rats were treated with an antagonist specific to EDNRA (BQ123) or EDNRB (BQ788), or when mice were treated with BQ123, BQ788 or BQ123 + BQ788. In conclusion, endothelin peptide is produced before ovulation and the contractile action of EDN2 within the ovary is facilitated via EDNRA. In addition, findings of this study indicate synergistic interactions among contractile factors affect ovulatory outcome, while the role of EDNRB alone in the process of ovulation requires further investigation.

Identification of a novel role for endothelins within the oviduct.

Endothelins were first identified as potent vasoactive peptides; however, diversity in the biological function of these hormones is now evident. We have identified a novel role for endothelins: a requirement for these peptides within the oviduct during fertilization and/or early embryo development. In vivo, treatment after ovulation with a dual endothelin receptor antagonist (tezosentan) decreased the number of two-cell embryos that could be collected from within the oviducts. In vitro fertilization experiments showed that gamete viability and their ability to fertilize were not affected by treatment with this antagonist, suggesting that the effect observed in vivo was mediated by the oviduct itself. Expression of mRNA for all three isoforms of the endothelins and both receptor subtypes was detectable within the oviduct. Expression of mRNA for endothelin-3 was regulated by gonadotropins in epithelial cells of the oviduct and increased specifically within the isthmus of this structure. Immunostaining revealed localization of both endothelin receptors A and B to the columnar epithelial cells within the oviduct, suggestive of a local role for endothelins in the regulation of epithelial function and ultimately oviductal secretions. A microarray analysis revealed three likely endothelin-regulated protein networks for future analysis: the TGFbeta, IL-10, and CCAAT/enhancer-binding protein superfamilies. Overall, these results suggest a novel and requisite role for endothelins within the oviduct during fertilization and/or early embryo development.

Kalirin is under-expressed in Alzheimer's disease hippocampus.

To identify genes aberrantly expressed in the brain of individuals with Alzheimer's Disease (AD), we analyzed RNA extracts from the hippocampus and cerebellum from 19 AD patients and 15 age- and sex-matched control subjects. Our analysis identified a number of genes that were over-expressed or under-expressed specifically in AD hippocampus. Among these genes, kalirin was the most consistently under-expressed in AD hippocampus, which was verified by semi-quantitative RT-PCR and real time PCR. Kalirin is predominantly expressed in the brain, particularly in the hippocampus, and plays crucial roles in neuronal stability and growth. Our observation is the first to relate kalirin to AD and a human disease. In addition to kalirin, the genes for voltage-gated Ca++ channel gamma subunit 3 and visinin-like protein 1 (a Ca++ sensor protein) were under-expressed, whereas inositol 1,4,5-triphosphate 3-kinase B was over-expressed in AD hippocampus. Collectively, these differential expressions could severely impair calcium homeostasis. Remarkably, these aberrant gene expressions in AD hippocampus were not observed in AD cerebellum. Furthermore, housekeeping genes such as ribosomal protein genes are not affected by AD. These results provide new insights into the biochemistry of AD.

Trans-activation, cis-activation and signal selection of gonadotropin receptors.

It has been thought that when a hormone binds to a receptor, the liganded receptor activates itself and generates hormone signals, such as the cAMP signal and the inositol phosphate signal (cis-activation). We describe that a liganded LH receptor or FSH receptor molecule is capable of intermolecularly activating nonliganded receptors (trans-activation). Particularly, intriguing is the possibility that a pair of compound heterozygous mutants, one defective in binding and the other defective in signaling, may cooperate and rescue signaling. Furthermore, trans-activation of the binding deficient receptors examined in our studies generates either the cAMP signal or the IP signal, but not both. Trans-activation and selective signal generation have broad implications on signal generation mechanisms, and suggest new therapeutic approaches.

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.

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.

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.