IDENTIFICATION AND CHARACTERIZATION OF CONTACT SITES BETWEEN HUMAN CHORIONIC GONADOTROPIN AND LUTEINIZING HORMONE/CHORIOGONADOTROPIN RECEPTOR

The luteinizing hormone receptor (LHR) belongs to the G protein-coupled receptorfamily. It consists of two distinct domains; the N-terminal extracellular exodomain and themembrane associated endodomain which includes 7 transmembrane domains, 3 exoloops, 3cytoloops and a C-terminal tail. Sequence alignment and computer modeling suggest thepresence of Leu Rich Repeat (LRR) motifs in the exodomain. Although their structuralsimilarity is high, each LRR is not equally important for hormone binding. Ala-scanning andtruncation studies performed in our laboratory suggest that LRR2 and LRR4 appear to be themost crucial. The Ala-scanning data suggest that Leu103 and Ile105 in LRR4 are important forhormone binding. However, it is not clear whether these two residues make direct contact withhuman chorionic gonadotropin (hCG) or if they are necessary for the overall structural integrityof LRR4. In this work, the LHR peptide mimics of LRR4 were used for photoaffinity labeling todetermine whether Leu103 and Ile105 directly interact with hormone. Furthermore, LRR4peptides containing the photoactivable benzoylphenylalanine (Bpa) were used to determinewhether the LRR structure really exists in the LHR exodomain, whether LRR 4 interact withhCG, and which residues of LRR4 interact with hCG. Bpa was directly incorporated intodifferent positions of the LRR4 peptide sequence to examine the labeling ability of individualamino acids. The results suggest that LRR4, in particular the sequence of Lys101-Cys106,makes direct contact with hCG. However Leu103 and Ile105 do not interact with hCG but mayform the hydrophobic core of the LRR4 loop, which appears to be crucial for the LRR structure.Existing data suggest that glycoprotein hormones initially bind the exodomain. Thehormone/exodomain complex undergoes conformational adjustments and stimulates theendodomain of the receptor to generate hormone signals. The exoloops modulate hormonebinding and signaling; however, little is known about whether the hormone/exodomain complexcontacts the endodomain. To address this issue, we investigated whether the exoloops interactwith the hormone. First, we examined exoloop 3 that connects transmembrane domains 6 and 7which are important for signal generation. We present the first physical evidence that LHRexoloop 3 interacts with hCG

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

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 \u3c 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

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

Functional Integration of the Conserved Domains of Shoc2 Scaffold

<div><p>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.</p></div

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 binding of Raf-1 in 293FT cells.

<p><b><i>A.</i></b> Schematic representation of the full-length and truncated Shoc2-tRFP constructs. <b><i>B</i></b><i>.</i> 293FT cells were transiently co-transfected with expression vectors encoding full-length or truncated tagRFP-tagged Shoc2 and YFP-Raf-1 or YFP-M-RAS. Thirty-six hours post-transfection, cells were lysed. GST-Raf-1 was precipitated with glutathione-coupled beads. HA-M-Ras was immuno-precipitated (IP) with HA antibody. The precipitated fraction was analyzed by immuno-blotting (IB) with tRFP and subsequently with Raf-1 and HA-antibodies to detect Raf-1 and Ras. Cell lysates were immunoblotted with Raf-1 antibodies to monitor expression of GST-Raf-1, HA antibodies to monitor expression of HA-M-Ras proteins, and tRFP antibodies to monitor expression of Shoc2 and its corresponding mutant used in panel IP. Results in each panel are representative of three independent experiments.</p

Wild-type Shoc2 and Shoc2 truncated mutants do not have a dominant-interfering effect in Cos-NT cells.

<p><b><i>A</i></b>, Cos1 cells were transiently transfected with full-length Shoc2-tRFP or Shoc2-tRFP truncated mutants. Cells were serum-starved for up to 12 hours and treated with 0.2 ng/ml EGF for indicated times at 37°C. The lysates were probed by immuno-blotting (IB) for Shoc2, tRFP, activated ERK1/2 (pERK1/2), total ERK1/2 (tERK1/2) and GAPDH (loading control). Dotted line shows area of the blot that was cropped to minimize occupied space. * denotes a proteolytic fragment of Shoc2-tRFP that is detected by immuno-blotting (IB) in cells expressing full-length Shoc2-tRFP. <b><i>B</i></b>, Multiple blots from the experiments exemplified in <b><i>A</i></b> were analyzed. Bars represent the mean values (±S.E., <i>n</i> = 4) of phosphorylated ERK1/2 activity normalized to total ERK1/2 in arbitrary units (pERK1/2/ERK), P = 0.938 (one-way ANOVA test was used to determine differences in phosphorylated ERK1/2 activity).</p

Defining the LRRs of Shoc2.

<p><b><i>A</i></b><b>,</b> Protein sequences from twenty two Shoc2 orthologues were utilized to generate an alignment of full-length Shoc2. The alignment was use to define the percent of similarity and identity of each Shoc2 orthologue to human Shoc2. <b><i>B</i></b><b>,</b> Protein sequences from ten Shoc2 vertebrate orthologues were utilized to generate an alignment of the N-terminal domain. The alignment was use to define the percent of identity of each Shoc2 orthologue to human Shoc2. <b><i>C</i></b>, Schematic representation of Shoc2 LRR and non-LRR regions. <b><i>D,</i></b> Multiple alignment of the individual twenty one LRRs of human Shoc2. The motif includes conserved sequence positions for the LRRs. Residue conservation color scheme: residues boxed in black are identical, and those boxed is dark and light grey are conserved substitutions. <b><i>E</i></b>, Ribbon representation of the model structure of Shoc2. Three structure elements characterize the fold of this protein: tandemly repeating units – LRRs, the β sheets that occupy the concave face of the scaffold and a regular array of possible helixes that characterizes its convex face.</p

Shoc2 interaction with M-Ras and Raf-1.

<p><b><i>A,</i></b> 293FT cells transiently co-transfected with expression vectors encoding full-length Shoc2-tRFP, YFP-Raf-1 or YFP-M-RAS. Twenty-four hours post-transfection, cells were starved for 16 hours, stimulated with EGF (2 ng/ml) for 15min and lysed. YFP-Raf-1 or YPF-M-Ras were immunoprecipitated (IP) using GFP antibody, and the immunoprecipitates were probed by immuno-blotting (IB) with Shoc2 and, subsequently, with GFP- and M-Ras antibodies to detect Raf-1 and Ras. Cell lysates were first immunoblotted with anti-Shoc2 antibodies to monitor Shoc2 expression, and then immunoblotted with anti-Raf-1 antibodies to monitor expression of YFP-Raf-1 and M-Ras antibody to monitor expression of YFP-M-Ras proteins. * denotes a proteolytic fragment of Shoc2-tRFP that is often detected by IB in cells expressing full-length Shoc2-tRFP. Shoc2-tRFP signal that is visible on the Raf-1 blot is indicated. <b><i>B.</i></b> 293FT cells transiently expressing HA-M-Ras and HA-M-Ras mutants (S27N and G22V). Thirty-six hours post-transfection, cells were lysed. M-Ras was immunoprecipitated using HA antibody, and the immunoprecipitates were probed by IB with Shoc2 and, subsequently, with HA-antibodies to detect Raf-1 and Ras. Cell lysates were immunoblotted with anti-HA antibody to monitor expression of corresponding M-Ras mutants used in panel IP or Shoc2 Abs to monitor expression of endogenous Shoc2. <b><i>C</i></b>. Cos-SR cells transiently expressing HA-M-Ras and its mutants were subjected to IP as in <b><i>B</i></b>. M-Ras was immunoprecipitated using HA antibody, and the immunoprecipitates were probed by IB with Shoc2 and, subsequently, with HA-antibodies to detect M-Ras. Cell lysates were immunoblotted with anti-HA antibody to monitor expression of corresponding M-Ras mutants used in panel IP or tRFP Abs to monitor expression of Shoc2-tRFP. Results in each panel are representative of three independent experiments.</p