Effect of FSH Receptor‐Binding Inhibitor‐8 on FSH‐Mediated Granulosa Cell Signaling and Proliferation

Follicle‐stimulating hormone is important for mammalian reproduction. It acts through specific receptors located on the plasma membrane of granulosa cells in ovaries and Sertoli cells in testes. The binding of follicle‐stimulating hormone to its receptor activates intracytoplasmic signaling pathways leading to steroidogenesis. These steroids in turn regulate the follicle‐stimulating hormone action from the anterior pituitary through exerting negative feedback effect. In addition to steroids, non‐steroidal factors secreted by the ovaries are believed to modulate follicle‐stimulating hormone action through autocrine/paracrine mode. One such low molecular weight peptide referred to as follicle‐stimulating hormone receptor‐binding inhibitor‐8 purified from human follicular fluid has been extensively studied. Follicle‐stimulating hormone receptor‐binding inhibitor‐8 has been shown to inhibit binding of follicle‐stimulating hormone to its receptor. The present article describes the effect of follicle‐stimulating hormone receptor‐binding inhibitor‐8 on follicle‐stimulating hormone‐induced signaling in rat granulosa cells. Follicle‐stimulating hormone receptor‐binding inhibitor‐8 inhibited the follicle‐stimulating hormone‐induced cAMP, and the effect was observed to be mediated through the protein kinase A. Further, an inhibitory effect of follicle‐stimulating hormone receptor‐binding inhibitor‐8 on the granulosa cell proliferation was evaluated using COV434 cell line which is derived from the human granulosa cell tumor. The effect of the peptide on the cell cycle analysis showed an increase in apoptotic population and the arrest of G1 phase. These findings suggest that follicle‐stimulating hormone receptor‐binding inhibitor‐8 acts as a follicle‐stimulating hormone antagonist and affects the follicle‐stimulating hormone‐mediated signaling and proliferation in the granulosa cells.     

Design and Synthesis of Potent Bivalent Peptide Agonists Targeting the EphA2 Receptor

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Studies on the carboxyl terminal peptides of human seminal plasma inhibin (HSPI)

Observation of contradictory results with the in vitro assays for inhibin-like activity of the carboxyl terminal 28 amino acid peptide 67–94 with a disulfide loop, of human seminal plasma inhibin (HSPI), prompted us to synthesize both the linear and the cyclic peptides and test their ability to suppress the circulating levels of follicle stimulating hormone (FSH) in vivo in adult male rats. The linear peptide [Cys(Acm)^73,87] 67–94 of HSPI was synthesized by solid-phase peptide synthesis using fluorenylmethyloxycarbonyl (Fmoc) chemistry and a continuous-flow technology. The peptide was cyclized by direct iodine oxidation of the S-diacetamidomethyl peptide in dilute solution. In the in vivo assay the linear peptide did not affect the levels of FSH, whereas the cyclic peptide suppressed the levels of FSH significantly. Thus, the carboxyl terminal region of HSPI does have inhibin-like activity and perhaps has the active core of the protein.     

Inhibition of inositol monophosphatase (IMPase) at the calbindin-D28k binding site: Molecular and behavioral aspects

Bipolar-disorder (manic-depressive illness) is a severe chronic illness affecting ～1% of the adult population. It is treated with mood-stabilizers, the prototypic one being lithium-salts (lithium), but it has life threatening side-effects and a significant number of patients fail to respond. The lithium-inhibitable enzyme inositol-monophosphatase (IMPase) is one of the viable targets for lithium's mechanism of action. Calbindin-D28k (calbindin) up-regulates IMPase activity. The IMPase-calbindincomplex was modeled using the program MolFit. The in-silico model indicated that the 55–66 amino-acid segment of IMPase anchors calbindin via Lys59 and Lys61 with a glutamate in between (Lys–Glu–Lys motif) and that the motif interacts with residues Asp24 and Asp26 of calbindin. We found that differently from wildtype calbindin, IMPase was not activated by mutated calbindin in which Asp24 and Asp26 were replaced by alanine. Calbindin's effect was significantly reduced by a linear peptide with the sequence of amino acids 58–63 of IMPase (peptide 1) and by six amino-acid linear peptides including at least part of the Lys–Glu–Lys motif. The three amino-acid peptide Lys–Glu–Lys or five amino-acid linear peptides containing this motif were ineffective. Mice administered peptide 1 intracerebroventricularly exhibited a significant anti-depressant-like reduced immobility in the forced-swim test. Based on the sequence of peptide 1, and to potentially increase the peptide's stability, cyclic and linear pre-cyclic analog peptides were synthesized. One cyclic peptide and one linear pre-cyclic analog peptide inhibited calbindin-activated brain IMPase activity in-vitro. Our findings may lead to the development of molecules capable of inhibiting IMPase activity at an alternative site than that of lithium.     

Interaction of Follicle‐Stimulating Hormone (FSH) Receptor Binding Inhibitor‐8: A Novel FSH‐Binding Inhibitor,with FSH and its Receptor

Follicle‐stimulating hormone (FSH) receptor binding inhibitor (FRBI‐8) is a novel octapeptide purified from human ovarian follicular fluid. In vitro, it inhibits the binding of FSH to granulosa cells and in vivo, it induces atresia in developing follicles in rodents. This peptide, when administered to marmosets and bonnet monkeys, altered the circulating progesterone levels. This study was carried out to elucidate structure of the FRBI‐8 and understand its mechanism of inhibiting interaction of FSH to its receptors. Homology modeling predicted that the FRBI‐8 adopts a turn and random coil. This is further confirmed by circular dichroism and NMR. Docking studies of the FRBI‐8 with reported FSH–FSHR hormone binding (FSHR_HB) domain complex using zdock algorithm revealed that the FRBI‐8 binds to FSHβL2–FSHR_HB binding interface which is otherwise known to be crucial for activation of signal transduction cascade. FRBI‐8 analogs were designed by replacing the acidic amino acid residues at positions 2, 5 and 6 with Ala, individually. Docking studies revealed that D6A mutant (FRBI‐8^D6A) had a higher binding affinity than the native FRBI‐8. In vitro radioreceptor assay with FRBI‐8^D6A showed 50% lower IC₅₀ compared with the FRBI‐8, confirming the in silico observations. Thus, the study reveals that both FRBI‐8 and FRBI‐8^D6A interfered with the binding of FSH to its receptor.     

Determinants of corticotropin releasing factor. Receptor selectivity of corticotropin releasing factor related peptides

The corticotropin-releasing factor (CRF) peptide family is an important target in pharmaceutical research. The CRF system consists of two receptors, corticotropin releasing factor receptor 1 (CRF1R) and corticotropin releasing factor receptor 2 (CRF2R), a nonreceptor binding protein, and the peptide agonists of these receptors. The recent discovery of the CRF2R selective peptide agonists, UCN2, UCN3 and URP, prompted investigations into the structural source of CRF1R versus CRF2R selectivity of CRF peptide family members. Data from chimeric peptides demonstrated that amino acids in the N-terminus and C-terminus of CRF, UCN1, UCN2 and Sauvagine peptide families influence CRFR selectivity. Analysis of specific amino acid residues in the N-terminus and C-terminus demonstrated that the presence of a proline at position 11 and alanine at positions 35 and 39 (hCRF numbering) decreases CRF1R activity and increases CRF2R selectivity in CRF, UCN1 and sauvagine peptides. The availability of a large group of selective and nonselective CRF receptor peptide agonists will facilitate the development of CRF receptor selective drugs.     

Characterization of growth hormone releasing factor analog expression in Saccharomyces cerevisiae

An analog of growth hormone releasing factor (GRF), [Leu²⁷]GRF(1-40)-OH, has been expressed and secreted in Saccharomyces cerevisiae under the control of the α-factor gene promoter and prepro sequence. A single pair of consecutive basic residues served as a processing site between the a-factor sequences and the GRF sequences. [Leu²⁷]GRF(1-40)-OH from fermentor broth containing 20-30 mg/L of immuno-reactive peptides was shown to be correctly processed and to possess biological activity as measured in vitro and in vivo. Additional peptides purified from broth appear to result from proteolytic degradation of the original translation product. Analysis of the amino acid compositions and sequences of these peptides suggests that processing enzymes may be responsible for some of the degradation.     

Attempt to map the receptor binding sites of human follicle‐stimulating hormone using disulfide peptides of its β‐subunit indicates major involvement of the regions around disulfide bonds Cys28–Cys82 and Cys32–Cys84 in receptor binding of the hormone

Abstract: Follicle‐stimulating hormone (FSH) is a heterodimeric glycoprotein hormone secreted by the anterior pituitary. It plays a very important role in folliculogenesis in females and is responsible for spermatogenesis in males. The α‐subunit which is common within a species and the β‐subunit which is hormone‐specific are held together by noncovalent association. This association is very essential for the biological activity of the hormone. Each of these subunits are highly cross‐linked by disulfide bonds which appear to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This study was initiated to delineate the role of the disulfide bonds of hFSHβ in receptor binding of the hormone. Five intermolecular and one intramolecular disulfide peptides corresponding to the disulfide bonds found in hFSHβ were synthesized and screened along with their linear counterparts, for their ability to competitively inhibit the radiolabelled [¹²⁵I]hFSH from binding to the FSH receptor containing membranes from the testis of immature rats. The disulfide peptides Cys²⁸–Cys⁸² and Cys³²–Cys⁸⁴ were found to be the most potent in inhibiting radiolabelled hFSH from binding to its receptor. The results suggest the involvement of the regions around disulfide bonds Cys²⁸–Cys⁸² and Cys³²–Cys⁸⁴ in receptor binding of the hormone. The studies also suggest the involvement of βL2 and βL3 loop regions in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hFSH.     

Tritiated D-ala1-peptide T binding: A pharmacologic basis for the design of drugs which inhibit HIV receptor binding

The HIV virus initiates its infectious cycle through a high-affinity binding interaction between the envelope protein gp 120 and its receptor, the T4 (or CD4) molecule. An octapeptide sequence, termed peptide T, present in the second variable region of gp 120 from the ARV isolate, has been implicated as the attachement site. The core peptide required for activity has been further refined to a pentapeptide, and homologous pentapeptides are similarly positioned in 21 other sequenced HIV isolates. Utilizing a novel direct binding assay of ³H-D-ala¹-peptide T, we now report that synthetic peptides derived from these other isolates are potent competitors of peptide T binding to T4-positive lymphocytes and brain membranes. Direct peptide T binding is also competable by purified virion gp 120, indicating that these ligands are interactive at the same receptor. Peptide T has sequence relatedness to the peptide vasoactive intestinal peptide (VIP), and VIP and its relevant homologous pentapeptide, VIP[7–11], are also potent inhibitors of peptide T binding. To determine the essential structural features responsible for receptor activity we have studied a series of synthetic peptides substituted with single D-amino acid residues. These data reveal that the tyrosine of position 7 in peptide T, present in all natural viral isolates, is obligate for receptor activity. Structure/function analysis for a large number of analogs is presented. Significantly, this binding assay is highly correlated with peptide bioactivity in several independent systems, indicating that this methodology can be used for rapid screening of novel, potential anti-AIDS therapeutics whose target is inhibition of virus gpl20 binding.     

Probing the functional conformation of the tridecapeptide mating pheromone of Saccharomyces cerevisiae through study of disulfide-constrained analogs

Analogs of the Saccharomyces cerevisiaeα-mating factor, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, where Lys⁷ and Gln¹⁰ were replaced with Cys, Cys(CH₃), or Ser, were synthesized using solid-phase procedures on a phenylacetamidomethyl resin. Cyclo^7,10[Cys⁷,X⁹,Cys¹⁰,Nle¹²]α-factor, where X = D-Val, D-Ala, l -Ala and Gly, were prepared by on-resin cyclization using thallic trifluoroacetate in yields of 20–30%. Linear sulfhydryl-containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys⁷ and Gln¹⁰ with a cysteine residue resulted in an over 100-fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5–10-fold more active than their sulfhydryl-contaihing homologs, and cyclo^7,10[Cys⁷,L-Ala⁹,Cys¹⁰,Nle¹²] α-factor was 50-fold more potent than linear analogs containing Ser or Cys(CH₃) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the α-factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5-aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form β-turns spanning residues 7–10 than their less active linear counterparts. The results provide strong evidence that this β-turn is important for optimal signal transduction by α-factor.     

Conformational study of the neurotensin and Substance P fragments: Pro-Arg-Arg-Pro and Arg-Pro-Lys-Pro

The conformational behaviour of the basic hydrophilic Pro-Arg-Arg-Pro and Arg-Pro-Lys-Pro peptides, neurotensin (NT) and Substance P fragments, has been taken up by semi-empirical calculations. The presence of two Pro residues prevents these peptides from giving any folded structure (α helix, β turn.). In both peptides the most stable conformations are essentially relative to more or less stretched structures; structures involving one or more residues in a γ turn form are often encountered in Pro-Arg-Arg-Pro peptide while mixed structures involving residues in very different conformations are found for the Arg-Pro-Lys-Pro-peptide. In both peptides, positively charged Lys and Arg side-chains most often point in opposite directions. The Pro-Arg-Arg-Pro peptide is part of the active NT (7–13) fragment where both Arg residues are necessary to the activity. A tentative study shows that the hydrophilic tetrapeptide induces NT (7–13) stretched conformations.     

The relationships between blood lead levels and serum follicle stimulating hormone and luteinizing hormone in the National Health and Nutrition Examination Survey 1999-2002

The relationships between blood lead levels and serum follicle stimulating hormone and luteinizing hormone were assessed in a nationally representative sample of women, 35-60 years old, from the National Health and Nutrition Examination Survey 1999-2002. The blood lead levels of the women ranged from 0.2 to 17.0 μg/dL. The estimated geometric mean was 1.4 μg/dL, and the estimated arithmetic mean was 1.6 μg/dL. As the blood lead level increased, the concentration of serum follicle stimulating hormone increased in post-menopausal women, women who had both ovaries removed, and pre-menopausal women. The concentration of luteinizing hormone increased as blood lead level increased in post-menopausal women and women who had both ovaries removed. The lowest concentrations of blood lead at which a relationship was detected were 0.9 μg/dL for follicle stimulating hormone and 3.2 μg/dL for luteinizing hormone. Lead may act directly or indirectly at ovarian and non-ovarian sites to increase the concentrations of follicle stimulating hormone and luteinizing hormone.     

Anticancer activity of VmCT1 analogs against MCF‐7 cells

Antimicrobial peptides are considered promising drug candidates due to their broad range of activity. VmCT1 (Phe–Leu–Gly–Ala–Leu–Trp–Asn–Val–Ala–Lys–Ser–Val–Phe–NH₂) is an α‐helical antimicrobial peptide that was obtained from the Vaejovis mexicanus smithi scorpion venom. Some of its analogs showed to be as antimicrobial as the wild type, and they were designed for understanding the influence of physiochemical parameters on antimicrobial and hemolytic activity. Some cationic antimicrobial peptides exhibit anticancer activity so VmCT1 analogs were tested to verify the anticancer activity of this family of peptides. The analogs were synthesized, purified, characterized, and the conformational studies were performed. The anticancer activity was assessed against MCF‐7 mammary cancer cells. The results indicated that [Glu]⁷‐VmCT1‐NH₂, [Lys]³‐VmCT1‐NH₂, and [Lys]⁷‐VmCT1‐NH₂ analogs presented moderated helical tendency (0.23–0.61) and tendency of anticancer activity at 25 μmol/L in 24 hr of experiment; and [Trp]⁹‐VmCT1‐NH₂ analog that presented low helical tendency and moderated anticancer activity at 50 μmol/L. These results demonstrated that single substitutions on VmCT1 led to different physicochemical features and could assist on the understanding of anticancer activity of this peptide family.     

The entire γ-carboxyglutamic acid- and helical stack-domains of human coagulation factor IX are required for optimal binding to its endothelial cell receptor

The minimal region of the γ-carboxyglutamic acid (Gla) domain of human factor (f) IX that interacted with its putative bovine aortic endothelial cell (BAEC) receptor was examined by chemical synthesis of peptides with sequence counterparts in this region of the protein, and assessment of their relative abilities to compete with fIX for receptor binding. We found that IC₅₀ values (total peptide concentrations needed to achieve 50% inhibition of binding of [¹²⁵I]-fIX to BAEC) were ca. 18 nM for unlabeled fIX and 23 nM for the peptide consisting of the entire Gla domain/helical stack (HS) region (residues 1–47) of fIX. The peptide containing only the Gla domain of fIX (residues 1-38) displayed an IC⁵⁰ value of >500 nM for this same competitive binding, whereas peptides containing sequences present in positions 1–14 and 1–24 of the Gla domain of human fIX did not significantly compete with [¹²⁵I]-fIX for BAEC binding. We conclude that whereas a specific receptor recognition element is present within residues 1–14 of fIX, as has previously been concluded by others and by us, full expression of this epitope requires its presence within the entire Gla domain and HS for proper folding. All determinants for proper folding of fIX that lead to BAEC receptor binding appear to be present within these two domains. © Munksgaard 1996.     

Construction and activity of a novel GHRH analog, Pro-Pro-hGHRH（1-44）-Gly-Gly-Cys

AIM: To construct another growth hormone releasing hormone (GHRH) analog, Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys peptide and to compare its activity with that of Pro-Pro-hGHRH(1-44)OH peptide. METHODS: The pro-pro-hGHRH(1-44)-gly-gly-cys DNA fragment was synthesized by polymerase chain reaction. The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3). The Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys peptide was purified to homogeneity by cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25 and Sephadex G-25 column chromatography. The peptide molecular mass was determined by electrospray ionization mass spectroscopy (ESI-MS). Its purity was determined by SDS-PAGE. The concentration of growth hormone (GH) stimulated by GHRH and its analogs was determined with antiserum kit against human GH. The other human hormones as hTSH, hFSH, hLH, and hPRL were determined with a paramagnetic particle chemiluminescent immunoassay kit. RESULTS: The molecular weight of Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys was 5455.4 kDa which was coincident with the theoretical calculations. All the three peptides at 5 mg/L stimulated GH release from the human fetal pituitary but only the difference between Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys group and blank group was significant (P<0.05). Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys 0.01, 0.1, and 1 mg/L and Pro-Pro-hGHRH (1-44)OH 0.1 and 1 mg/L stimulated GH release from rat pituitary in a concentration-dependent manner (P<0.05, P<0.01). At the same concentration Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys stimulated more GH release than Pro-Pro-hGHRH (1-44)OH. Pro-Pro-hGHRH (1-44)OH 0.01 mg/L and hGHRH (1-40)OH 2 mg/L did not stimulate GH release from rat pituitary (P>0.05). Pro-Pro-hGHRH (1-44)OH and Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys peptides at 5 mg/L did not stimulate hTSH, hFSH, hLH, and hPRL release from human fetal pituitary in vitro. CONCLUSION: Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys had a better activity than that of Pro-Pro-hGHRH(1-44)OH. Variation at C-terminus of GHRH could modulate its GH releasing activity.     

Structure–activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin

Abstract: Peptides derived from the third α‐helix of the homeodomain (residues 43–58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N‐ and C‐terminally truncated peptides, as well as a series of alanine substitution analogues, were studied. Cell penetration assays using human cell cultures with these peptides revealed that the C‐terminal segment ⁵²Arg‐Arg‐Met‐Lys‐Trp‐Lys‐Lys⁵⁸ of the parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide’s basic residues led to markedly decreased cell internalization ability, whereas replacement of hydrophobic residues was tolerated surprisingly well. Subcellular localization was seen to be affected by substitutions, with analogues being addressed preferentially to the cytosol or to the nucleus. Conformational constriction of the Penetratin sequence through placement and oxidation of flanking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.     

Melanin concentrating hormone analogues: contraction of the cyclic structure. 1. Agonist activity

Melanin concentrating hormone (MCH) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, which is synthesized in the hypothalamus and secreted by the neurohypophysis of teleost fishes. This hormone exhibits both MCH-like as well as alpha-MSH (alpha-melanocyte stimulating hormone) like activity. We have examined the role of the disulfide bond for the two contrasting melanotropic activities of MCH. Nine analogues of the parent peptide were synthesized and characterized for biological activity. The disulfide ring was contracted from the 5-14 to the 7-14, 8-14, and 10-14 residues with concomitant substitution of alanine for Cys at position 5 in each of the heptadecapeptides. Similar substitutions were made in a series of MCH analogues. In addition, the following cyclic peptides also were synthesized: [Cys7]MCH, [Cys8]MCH, and [Cys10]MCH. The fish-skin bioassay is sensitive to MCH at a concentration of 10(-12) M. All ring-contracted analogues were inactive at 10(-6) M or lower concentrations; less than 1/1,000,000 compared to MCH (1.0) except [Ala5,Cys8]MCH (0.0008; 1/1250), [Cys10]MCH (0.000 09; 1/10,000), and [Cys8]MCH (0.000 001; 1/1,000,000). In the frog-skin bioassay, [Ala5,Cys10]MCH, although lacking MCH-like activity in the fish-skin bioassay, was equipotent to MCH in its alpha-MSH-like component of activity. Most other analogues were either inactive or much less active than MCH in stimulating melanosome dispersion. These results demonstrate that the disulfide bond between positions 5 and 14 is essential for the MCH-like activity since contraction of the ring generally leads to inactive peptides. Contraction of the disulfide bridge does not, however, have as great an effect on the MSH-like activity of MCH.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys9−Cys57 and Cys23−Cys72 in receptor binding of the hormone

Abstract: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α‐ and β‐subunits of hCG are highly cross‐linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGβ in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGβ were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [¹²⁵I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9?57) was found to be ≈ 4‐fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23?72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys⁹?Cys⁵⁷ and Cys²³?Cys⁷² of the β‐subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Synthesis,conformation, receptor binding and biological activities of monobiotinylated human insulin‐like peptide 3*

Abstract: Biotin‐avidin immobilization has been routinely used as a tool to study peptide–receptor and peptide–antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides’ receptor, and to analyse ligand‐receptor binding. Insulin‐like peptide 3 (INSL3) is a peptide hormone which contains A‐ and B‐chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein‐coupled receptor, we chemically synthesized N^α‐mono‐biotinylated human INSL3 (B‐hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with ³³P‐labelled relaxin H2 (B₃₃). The modified B‐hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B‐hINSL3 contains a higher α‐helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N‐terminal region of the A‐chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N‐terminus of the A‐chain promoted conformational stability which, in turn, permitted better receptor activation.     

Secondary structure analysis of synthetic peptides of the extracellular domain of the human follicle‐stimulating hormone receptor

Abstract: Follicle‐stimulating hormone receptor (FSHR) is a glycoprotein hormone receptor and possesses a large extracellular domain (ECD) instrumental in hormone binding. The ECD is characterized by the presence of leucine‐rich repeat (LRR) structures made up of α‐helices flanked by β‐strands. Our previous studies with the synthetic peptides corresponding to the potentially surface‐oriented regions of the ECD had led to the identification of some of these regions in either FSH‐binding or FSH‐induced cAMP production or both. This study was undertaken with an aim to correlate the findings made in vitro with the secondary structures of the respective peptides. Accordingly, all peptides were screened for their secondary structures in different biochemical environments. This study correlates the observed α‐helical signature with the previously demonstrated activity in signal generation for peptides 15–31 and 216–235 hFSHR, while FSH binding is correlated with the maintenance of β‐sheet structure in peptides 285–300 and 297–310 hFSHR as observed in vitro.