Identification of an extracellular segment of the oxytocin receptor providing agonist-specific binding epitopes

2001 ◽  
Vol 354 (2) ◽  
pp. 465-472 ◽  
Author(s):  
Stuart R. HAWTIN ◽  
Helen C. HOWARD ◽  
Mark WHEATLEY
Keyword(s):  
Specific Binding ◽  
Large Family ◽  
Peptide Hormone ◽  
Distal Portion ◽  
Terminal Segment ◽  
Target Tissue ◽  
Agonist Binding ◽  
Unrelated Sequence ◽  
N Terminus ◽  
Vasopressin V1a Receptor

The effects of the peptide hormone oxytocin are mediated by oxytocin receptors (OTRs) expressed by the target tissue. The OTR is a member of the large family of G-protein-coupled receptors. Defining differences between the interaction of agonists and antagonists with the OTR at the molecular level is of fundamental importance, and is addressed in this study. Using truncated and chimaeric receptor constructs, we establish that a small 12-residue segment in the distal portion of the N-terminus of the human OTR provides important epitopes which are required for agonist binding. In contrast, this segment does not contribute to the binding site for antagonists, whether peptide or non-peptide. It does, however, have a role in agonist-induced OTR signalling. Oxytocin is also an agonist at the vasopressin V1a receptor (V1aR). A chimaeric receptor (V1aRN-OTR) was engineered in which the N-terminus of the OTR was substituted by the corresponding, but unrelated, sequence from the N-terminus of the V1aR. We show that the V1aR N-terminus present in V1aRN-OTR fully restored both agonist binding and intracellular signalling to a dysfunctional truncated OTR construct. The N-terminal segment does not, however, contribute to receptor-selective agonism between the OTR and the V1aR. Our data establish a key role for the distal N-terminus of the OTR in providing agonist-specific binding epitopes.

Agonist binding to peptide hormone receptors

2003 ◽  
Vol 31 (1) ◽  
pp. 35-39 ◽  
Author(s):  
M. Wheatley ◽  
S.R. Hawtin ◽  
V.J. Wesley ◽  
H.C. Howard ◽  
J. Simms ◽  
...  
Keyword(s):  
Hormone Receptors ◽  
Specific Binding ◽  
Peptide Hormone ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Receptor Interaction ◽  
Agonist Binding ◽  
Absolute Requirement ◽  
N Terminus ◽  
Peptide Hormone Receptors

A fundamental issue in molecular pharmacology is to define how agonist–receptor interaction differs from that of antagonist–receptor interaction. The V1a vasopressin receptor (V1aR) is a member of a family of related G-protein-coupled receptors (GPCRs) that are activated by vasopressin, oxytocin (OT) and related peptides. A segment of the N-terminus that was required for agonist binding, but not antagonist binding, was identified by characterizing truncated V1aR constructs. Site-directed mutagenesis revealed that a single residue (Arg46) was critical for agonist binding and receptor activation. The N-terminus of the related OT receptor (OTR) could recover agonist binding in a chimaeric OTRN–V1aR construct. Furthermore, Arg34 of the human OTR, which corresponds to Arg46 of the rat V1aR, provided agonist-specific binding epitopes in the OTR, indicating a conserved function of this locus throughout this GPCR subfamily. Mutation of Arg46 revealed that high-affinity agonist binding had an absolute requirement for arginine at this position.

Metallo-oxidase Enzymes: Design of their Active Sites

2011 ◽  
Vol 64 (3) ◽  
pp. 231 ◽  
Author(s):  
Zhiguang Xiao ◽  
Anthony G. Wedd
Keyword(s):  
Metal Ions ◽  
Active Sites ◽  
Specific Binding ◽  
Large Family ◽  
Outer Sphere ◽  
Transition Metal Ions ◽  
Substrate Oxidation ◽  
Cellular Functions ◽  
Domains Of Life ◽  
Low Valent

Multi-copper oxidases are a large family of enzymes prevalent in all three domains of life. They couple the one-electron oxidation of substrate to the four-electron reduction of dioxygen to water and feature at least four Cu atoms, traditionally divided into three sites: T1, T2, and (binuclear) T3. The T1 site catalyzes substrate oxidation while a trinuclear cluster (comprising combined T2 and T3 centres) catalyzes the reduction of dioxygen. Substrate oxidation at the T1 Cu site occurs via an outer-sphere mechanism and consequently substrate specificities are determined primarily by the nature of a substrate docking/oxidation (SDO) site associated with the T1 Cu centre. Many of these enzymes ‘moonlight’, i.e. display broad specificities towards many different substrates and may have multiple cellular functions. A sub-set are robust catalysts for the oxidation of low-valent transition metal ions such as FeII, CuI, and MnII and are termed ‘metallo-oxidases’. They play essential roles in nutrient metal uptake and homeostasis, with the ferroxidase ceruloplasmin being a prominent member. Their SDO sites are tailored to facilitate specific binding and facile oxidation of these low-valent metal ions and this is the focus of this review.

scholarly journals Identification of the angiotensin II receptor in rat mesenteric artery

1984 ◽  
Vol 223 (3) ◽  
pp. 659-671 ◽  
Author(s):  
J McQueen ◽  
G D Murray ◽  
P F Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Divalent Cations ◽  
Target Tissue ◽  
Angiotensin Ii Receptor ◽  
High Affinity ◽  
Rat Mesentery ◽  
Rat Mesenteric Artery

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

scholarly journals Selective Inactivation of Adrenomedullin over Calcitonin Gene-related Peptide Receptor Function by the Deletion of Amino Acids 14-20 of the Mouse Calcitonin-like Receptor

2004 ◽  
Vol 279 (19) ◽  
pp. 20387-20391 ◽  
Author(s):  
Daniela Koller ◽  
Lars M. Ittner ◽  
Roman Muff ◽  
Knut Husmann ◽  
Jan A. Fischer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Peptide Hormone ◽  
Receptor Function ◽  
Cgrp Receptor ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
N Terminus ◽  
Camp Formation

The receptors for the neuropeptide calcitonin (CT) gene-related peptide (CGRP) and the multifunctional peptide hormone adrenomedullin (AM) are calcitonin-like receptor (CLR)/receptor-activity-modifying protein (RAMP) 1 and CLR/RAMP2 heterodimers, respectively. Here, the amino acid sequence TRNKIMT, corresponding to the residues 14-20 of the N terminus of the mouse (m) CLR, was found to be required for a functional mCLR/RAMP2 AM receptor. The deletion of amino acids 14-20 (Δ14-20) or their substitution by alanine (14-20A) did not affect the heterodimerization of the mCLR with mRAMP1 or mRAMP2, and the levels of expression at the surface of transiently transfected COS-7 cells were not altered. In mRAMP1/mCLR- or mRAMP1/mCLR-(Δ14-20)-expressing cells CGRP stimulated cAMP formation with EC50values of 0.12 ± 0.01 and 1.5 ± 0.4 nm, respectively. In mRAMP2/mCLR-expressing cells the EC50of AM was 0.8 ± 0.2 nm. However, in cells expressing mRAMP2/mCLR-(Δ14-20) up to 10-6mAM failed to stimulate cAMP production. In mRAMP2/mCLR-(14-20A) expressing cells the cAMP response to AM was minimally restored, and the EC50was >100 nm. In conclusion, the deletion of the amino acid sequence TRNKIMT of the extreme N terminus of the mCLR maintained CGRP receptor function of mRAMP1/receptor heterodimers, but AM no longer activated the mutant mCLR-(Δ14-20) in the presence of mRAMP2. The TRNKIMT sequence is required for normal mCLR/mRAMP2 association, and as a consequence, high affinity AM binding signaling the activation of adenylyl cyclase.

scholarly journals Functional characterization of an N-terminally-truncated mitochondrial porin expressed in Neurospora crassa

2017 ◽  
Vol 63 (8) ◽  
pp. 730-738 ◽  
Author(s):  
Sabbir R. Shuvo ◽  
Uliana Kovaltchouk ◽  
Abdullah Zubaer ◽  
Ayush Kumar ◽  
William A.T. Summers ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Outer Membrane ◽  
Functional Characterization ◽  
Helical Structure ◽  
Terminal Segment ◽  
Wild Type ◽  
Mitochondrial Porin ◽  
N Terminus

Mitochondrial porin, which forms voltage-dependent anion-selective channels (VDAC) in the outer membrane, can be folded into a 19-β-stranded barrel. The N terminus of the protein is external to the barrel and contains α-helical structure. Targeted modifications of the N-terminal region have been assessed in artificial membranes, leading to different models for gating in vitro. However, the in vivo requirements for gating and the N-terminal segment of porin are less well-understood. Using Neurospora crassa porin as a model, the effects of a partial deletion of the N-terminal segment were investigated. The protein, ΔN2-12porin, is assembled into the outer membrane, albeit at lower levels than the wild-type protein. The resulting strain displays electron transport chain deficiencies, concomitant expression of alternative oxidase, and decreased growth rates. Nonetheless, its mitochondrial genome does not contain any significant mutations. Most of the genes that are expressed in high levels in porin-less N. crassa are expressed at levels similar to that of wild type or are slightly increased in ΔN2-12porin strains. Thus, although the N-terminal segment of VDAC is required for complete function in vivo, low levels of a protein lacking part of the N terminus are able to rescue some of the defects associated with the absence of porin.

scholarly journals Mechanistic basis for the activation of plant membrane receptor kinases by SERK-family coreceptors

2018 ◽  
Vol 115 (13) ◽  
pp. 3488-3493 ◽  
Author(s):  
Ulrich Hohmann ◽  
Julia Santiago ◽  
Joël Nicolet ◽  
Vilde Olsson ◽  
Fabio M. Spiga ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Specific Binding ◽  
Peptide Hormone ◽  
Receptor Activation ◽  
Kinase Domain ◽  
Membrane Receptor ◽  
Activation Mechanism ◽  
In Planta ◽  
Affinity Ligand ◽  
Receptor Kinases

Plant-unique membrane receptor kinases with leucine-rich repeat ectodomains (LRR-RKs) can sense small molecule, peptide, and protein ligands. Many LRR-RKs require SERK-family coreceptor kinases for high-affinity ligand binding and receptor activation. How one coreceptor can contribute to the specific binding of distinct ligands and activation of different LRR-RKs is poorly understood. Here we quantitatively analyze the contribution of SERK3 to ligand binding and activation of the brassinosteroid receptor BRI1 and the peptide hormone receptor HAESA. We show that while the isolated receptors sense their respective ligands with drastically different binding affinities, the SERK3 ectodomain binds the ligand-associated receptors with very similar binding kinetics. We identify residues in the SERK3 N-terminal capping domain, which allow for selective steroid and peptide hormone recognition. In contrast, residues in the SERK3 LRR core form a second, constitutive receptor–coreceptor interface. Genetic analyses of protein chimera between BRI1 and SERK3 define that signaling-competent complexes are formed by receptor–coreceptor heteromerization in planta. A functional BRI1–HAESA chimera suggests that the receptor activation mechanism is conserved among different LRR-RKs, and that their signaling specificity is encoded in the kinase domain of the receptor. Our work pinpoints the relative contributions of receptor, ligand, and coreceptor to the formation and activation of SERK-dependent LRR-RK signaling complexes regulating plant growth and development.

OESTROGEN RECEPTORS IN HUMAN UTERINE TISSUE

1971 ◽  
Vol 50 (2) ◽  
pp. 209-229 ◽  
Author(s):  
L. H. EVANS ◽  
R. HÄHNEL
Keyword(s):  
Human Tissue ◽  
Specific Binding ◽  
Binding Activity ◽  
Human Myometrium ◽  
Target Tissue ◽  
Uterine Tissue ◽  
Tissue Slices ◽  
Binding Characteristics ◽  
Significant Difference

SUMMARY The uptake and retention of [3H]oestradiol-17β by human uterine and muscle tissue was studied in vitro by incubation of tissue slices. The binding characteristics of immature rat and adult human tissue were compared. Human endometrium had a greater affinity for tritiated oestradiol than human myometrium. Human uterine tissues bound less [3H]oestradiol-17β than rat uterine horns. The difference in retention between a target and non-target tissue in the human tissue was much smaller than in the rat, although still highly significant. Binding was inhibited by low concentrations of oestradiol-17β, U-11100A and hexoestrol, but was not inhibited by the same concentrations of oestrone, oestriol or oestradiol-17α. Freezing the uterine tissue resulted in a loss of specific binding activity. Binding was shown to vary with different uterine specimens. There was a significant difference in [3H]oestradiol-17β retention between the proliferative and secretory phases of the menstrual cycle. The highest endometrial retention was found in endometrial carcinoma.

BINDING OF LUTEINIZING HORMONE AND HUMAN CHORIONIC GONADOTROPHIN TO OVARIAN CELLS AND ACTIVATION OF ADENYLATE CYCLASE

1974 ◽  
Vol 61 (2) ◽  
pp. 179-191 ◽  
Author(s):  
Y. KOCH ◽  
U. ZOR ◽  
PRAKONG CHOBSIENG ◽  
S. A. LAMPRECHT ◽  
S. POMERANTZ ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Adenylate Cyclase ◽  
Specific Binding ◽  
Ovarian Tissue ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Target Tissue ◽  
Ovarian Cells ◽  
Receptor Sites ◽  
Effect Binding

SUMMARY The binding of 125I-labelled ovine luteinizing hormone (OLH) and human chorionic gonadotrophin (HCG) to unbroken cells or homogenates from rat and bovine ovaries was studied in relation to the hormonal activation of adenylate cyclase. Both hormones are preferentially bound to ovarian tissue (luteal as well as follicular), while adrenal, kidney and liver showed no specific binding. The bound HCG was associated chiefly with the fraction of the homogenate sedimenting at 1000 g. Addition of excess unlabelled hormone (HCG, OLH) together with the corresponding or heterologous 125I-labelled hormone reduced the radioactivity bound to the tissue by 70–90%; rat LH also competed with OLH and HCG for binding sites, but follicle-stimulating hormone, prolactin, growth hormone, adrenocorticotrophin or glucagon were without effect. Binding could be dissociated from activation of adenylate cyclase by a two-stage incubation: at 4 °C rat ovarian slices bound the labelled hormones, though binding efficiency was reduced to about 20%, but no stimulation of the enzyme was observed. Reincubation of these slices, after washing, at 37 °C in hormone-free medium resulted in maximal activation of adenylate cyclase. Addition of homologous antibodies before the second incubation step abolished the effect of OLH but not that of HCG on the activation of the enzyme. Similarly, addition of homologous antibodies or of excess unlabelled hormone caused dissociation of bound 125I-labelled OLH but not of 125I-labelled HCG from receptor sites. It is concluded that (i) HCG, OLH and rat LH are bound to the same receptor on ovarian cells, but binding of OLH to the receptor is more labile than that of HCG; (ii) the receptor appears to be located on the plasma membrane, since it was largely associated with the 1000 g pellet and since bound OLH remained accessible to antibody; (iii) activation of adenylate cyclase requires the continued occupation of the binding site by the hormone, but occupation of only a fraction of the receptor sites by hormone is adequate to induce maximal production of cyclic AMP by the target tissue.

Structure of the Sgt2 dimerization domain complexed with the Get5 UBL domain involved in the targeting of tail-anchored membrane proteins to the endoplasmic reticulum

2013 ◽  
Vol 69 (10) ◽  
pp. 2081-2090 ◽  
Author(s):  
Jung-Yu Tung ◽  
Yi-Chuan Li ◽  
Tai-Wen Lin ◽  
Chwan-Deng Hsiao
Keyword(s):  
Endoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Specific Binding ◽  
Tetratricopeptide Repeat ◽  
Dimerization Domain ◽  
Hydrophobic Residues ◽  
N Terminus ◽  
Tetratricopeptide Repeat Protein ◽  
Ta Proteins ◽  
Dual Functions

The insertion of tail-anchored membrane (TA) proteins into the appropriate membrane is a post-translational event that requires stabilization of the transmembrane domain and targeting to the proper destination. Sgt2, a small glutamine-rich tetratricopeptide-repeat protein, is a heat-shock protein cognate (HSC) co-chaperone that preferentially binds endoplasmic reticulum-destined TA proteins and directs them to the GET pathwayviaGet4 and Get5. The N-terminal domain of Sgt2 seems to exert dual functions. It mediates Get5 interaction and allows substrate delivery to Get3. Following the N-terminus of Get5 is a ubiquitin-like (Ubl) domain that interacts with the N-terminus of Sgt2. Here, the crystal structure of the Sgt2 dimerization domain complexed with the Get5 Ubl domain (Sgt2N–Get5Ubl) is reported. This complex reveals an intimate interaction between one Sgt2 dimer and one Get5 monomer. This research further demonstrates that hydrophobic residues from both Sgt2 and Get5 play an important role in cell survival under heat stress. This study provides detailed molecular insights into the specific binding of this GET-pathway complex.

Peptide Hormone “Receptors”: Specific Binding of3H-FSH to Testis

Endocrinology ◽  
1972 ◽  
Vol 90 (1) ◽  
pp. 39-46 ◽  
Author(s):  
ANTHONY R. MEANS ◽  
JUDITH VAITUKAITIS
Keyword(s):  
Hormone Receptors ◽  
Specific Binding ◽  
Peptide Hormone ◽  
Peptide Hormone Receptors
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