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1.
Residue 19 of the parathyroid hormone (PTH) has been shown to play an important role in both binding to and activation of the PTH receptor; specifically, Arg(19)-containing analogues have improved biological function over similar Glu(19) peptides [Shimizu et al. (2002) Biochemistry 41, 13224-13233]. Additionally the juxtamembrane portion of the receptor is involved in the different biological responses. Here, we determine the conformational preferences of PTH analogues to provide a structural basis for their biological actions. On the basis of circular dichroism results, the Arg(19) --> Glu(19) mutations within the context of both PTH(1-20) and PTH(1-34) analogues lead to increases in helix content, ranging from a 8-15% increase. High-resolution structures as determined by (1)H NMR and NOE-restrained molecular dynamics simulations clearly illustrate the difference between Arg(19) and Glu(19)-PTH(1-20), particularly with the extent and stability of the C-terminal helix. The Arg(19)-containing analogue has a well defined, stable alpha-helix from Ser(4)-Arg(19), while the Glu(19) analogue is less ordered at the C-terminus. On the basis of these observations, we propose that position 19 of PTH(1-20) must be alpha-helical for optimal interaction with the juxtamembrane portion of the receptor. This mode of binding extends the current view of PTH binding (indeed ligand binding for all class B GPCRs), which invokes a bihelical ligand with the C-terminus of the ligand interacting with the N-terminus of the receptor (responsible for binding) and the N-terminus of the ligand interacting with the seven-helical bundle (leading to receptor activation). 相似文献
2.
3.
Hawtin SR Wesley VJ Parslow RA Simms J Miles A McEwan K Wheatley M 《Molecular endocrinology (Baltimore, Md.)》2002,16(3):600-609
A fundamental issue in molecular endocrinology is to define how agonist:receptor interaction differs from antagonist:receptor interaction. The vasopressin V1a receptor (V1aR) is a member of a subfamily of related G protein-coupled receptors that are activated by the hormone AVP or related peptides. The N-terminus of the V1aR has recently been shown to be critical for binding agonists but not antagonists. Using a combination of N-terminally truncated constructs and alanine-scanning mutagenesis, individual residues that provide these agonist-specific binding epitopes have now been identified in this study. Our data establish that a single residue, Arg46, is critical for AVP binding to the V1aR. Systematic substitution revealed that Arg was required at this locus and could not be substituted by Lys, Glu, Leu, or Ala. In contrast, antagonist binding (cyclic or linear, peptide or nonpeptide) was unaffected. Disruption of Arg46 also resulted in defective intracellular signaling. Arginine is conserved at this locus in all members of the neurohypophysial peptide hormone receptor family cloned to date, indicative of a fundamental role in receptor function. In addition to Arg46, the residues Leu42, Gly43, Asp45 form a patch contributing to AVP binding. This study provides molecular insight into the role of the V1aR N-terminus and key differences between agonist and antagonist binding requirements. 相似文献
4.
Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) activate the PTH/PTHrP receptor to trigger parallel increases in adenylyl cyclase (AC) and phospholipase C (PLC). The amino (N)-terminal region of PTH-(1-34) is essential for AC activation. Ligand domains required for activation of PLC, PKC, and other effectors have been less well-defined, although some studies in rodent systems have identified a core region [hPTH-(29-32)] involved in PKC activation. To determine the critical ligand domain(s) for PLC activation, a series of truncated hPTH-(1-34) analogues were assessed using LLC-PK1 cells that stably express abundant transfected human or rat PTH/PTHrP receptors. Phospholipase C signaling and ligand-binding affinity were reduced by carboxyl (C)-terminal truncation of hPTH-(1-34) but were coordinately restored when a binding-enhancing substitution (Glu(19) --> Arg(19)) was placed within hPTH-(1-28), the shortest hPTH peptide that could fully activate both AC and PLC. Phospholipase C, but not AC, activity was reduced by substituting Gly(1) for Ser(1) in hPTH-(1-34) and was eliminated entirely by removing either residue 1 or the alpha-amino group alone. These changes did not alter binding affinity. These findings led to design of an analogue, [Gly(1),Arg(19)]hPTH-(1-28), that was markedly signal-selective, with full AC but no PLC activity. Thus, the extreme N-terminus of hPTH constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially hPTH-(28-31), contributes to PLC activation through effects upon receptor binding but is not required for full PLC activation. The N-terminal determinants of AC and PLC activation in hPTH-(1-34) overlap but are not identical, as subtle modifications in this region may dissociate activation of these two effectors. The [Gly(1),Arg(19)]hPTH-(1-28) analogue, in particular, should prove useful in dissociating AC- from PLC-dependent actions of PTH. 相似文献
5.
T J Gardella D Axelrod D Rubin H T Keutmann J T Potts H M Kronenberg S R Nussbaum 《The Journal of biological chemistry》1991,266(20):13141-13146
The first 4 residues of parathyroid hormone (PTH) are highly conserved in evolution and are important for biological activity. We randomly mutated codons 1-4 of human PTH (hPTH) with degenerate oligonucleotides and, after expression in COS cells, screened the mutants for receptor binding and cAMP-stimulating activity using ROS 17/2.8 cells. This survey identified Glu4 and Val2 as important determinants of receptor binding and activation, respectively. Positions 1 and 3 were more tolerant of substitutions indicating that these sites are less vital to hormone function. Activities of synthetic hPTH(1-34) analogs further demonstrated the importance of positions 2 and 4. The binding affinity of [Ala4,Tyr34] hPTH(1-34)NH2 was 100-fold reduced relative to [Tyr34]hPTH(1-34)NH2 (Kd values = 653 +/- 270 and 4 +/- 1 nM, respectively), and [Arg2, Tyr34]hPTH(1-34)NH2 was a weak partial agonist which bound well to the ROS cell receptor (Kd = 31 +/- 10 nM). The Arg2 analog was nearly as potent as PTH(3-34) as an in vitro PTH antagonist in osteoblast derived cells. However, unlike PTH(3-34), [Arg2]PTH was a full agonist in opossum kidney (OK) cells. These observations suggest that the activation domains of the OK and ROS cell PTH receptors are different. Thus, amino-terminal PTH analogs may be useful as probes for distinguishing properties of PTH receptors. 相似文献
6.
The effects of the peptide hormone oxytocin (OT) are mediated by the oxytocin receptor, which is a member of the G-protein-coupled receptor family. Defining differences between the binding of agonists and antagonists to the OTR, at the molecular level, is of fundamental importance to understanding OTR activation and to rational drug design. Previous reports have indicated that the N-terminus of the OTR is required for OT binding. The aim of this study was to identify which individual residues within the N-terminal domain of the human OTR provided these OT binding epitopes. A series of truncated OTRs and mutant receptor constructs with systematic alanine substitution were characterized with respect to their pharmacological profile and intracellular signaling capability. Although a number of residues within the OTR will be required for optimal OT-OTR interaction, our data establish that Arg(34) within the N-terminal domain contributes to high-affinity OT binding. Removal of Arg(34) by truncation or substitution resulted in a 2000-fold decrease in OT affinity. In addition, we show that the arginyl at this locus is required for high-affinity binding of agonists in general. However, the importance of Arg(34) is restricted to agonist interaction with the OTR, as it was not required for binding peptide antagonist or non-peptide antagonist. It is noteworthy that the corresponding Arg in the related rat V(1a) vasopressin receptor is also required for high-affinity agonist binding. This study defines, at the molecular level, the role of the N-terminus of the OTR in high-affinity agonist binding and identifies a key residue for this function. 相似文献
7.
The type 1 parathyroid hormore receptor (PTH1r) belongs to the class II family of G protein-coupled receptors. To delineate the sites in the PTH1r's N-terminal region, and the carboxy-core domain (transmembrane segments + extracellular loops) involved in PTH binding, we have evaluated the functional properties of 27 PTH1-secretin chimeras receptors stably expressed in HEK-293 cells. The wild type and chimeric receptors were analyzed for cell surface expression, binding for PTH and secretin, and functional responsiveness (cAMP induction) toward secretin and PTH. The expression levels of the chimeric receptors were comparable to that of the PTH1r (60-100%). The N-terminal region of PTH1r was divided into three segments that were replaced either singly or in various combinations with the homologous region of the secretin receptor (SECr). Substitution of the carboxy-terminal half (residues 105-186) of the N-terminal region of PTH1r for a SECr homologous segment did not reduced affinity for PTH but abolished signaling in response to PTH. This data indicate that receptor activation is dissociable from high affinity hormone binding in the PTH1r, and that the N-terminal region might play a critical role in the activation process. Further segment replacements in the N-termini focus on residues 105-186 and particularly residues 146-186 of PTH1r as providing critical segments for receptor activation. The data obtained suggest the existence of two distinct PTH binding sites in the PTH1r's N-terminal region: one site in the amino-terminal half (residues 1-62) (site 1) that participates in high-affinity PTH binding; and a second site of lower affinity constituted by amino acid residues scattered throughout the carboxy-terminal half (residues 105-186) (site 2). In the absence of PTH binding to site 1, higher concentrations of hormone are required to promote receptor activation. In addition, elimination of the interaction of PTH with site 2 results in a loss of signal transduction without loss of high-affinity PTH binding. Divers substitutions of the extracellular loops of the PTH1r highlight the differential role of the first- and third extracellular loop in the process of PTH1r activation after hormone binding. A chimera containing the entire extracellular domains of the PTH1r and the transmembrane + cytoplasmic domains of SECr had very low PTH binding affinity and did not signal in response to PTH. Further substitution of helix 5 of PTH1r in this chimera increased affinity for PTH that is close to the PTH affinity for the wild-type PTH1r but surprisingly, did not mediate signaling response. Additional substitutions of PTH1r's helices in various combinations emphasize the fundamental role of helix 3 and helix 6 on the activation process of the PTH1r. Overall, our studies demonstrated that several PTH1r domains contribute differentially to PTH binding affinity and signal transduction mechanism and highlight the role of the N-terminal domain and helix 3 and helix 6 on receptor activation. 相似文献
8.
Dean T Khatri A Potetinova Z Willick GE Gardella TJ 《The Journal of biological chemistry》2006,281(43):32485-32495
The principal receptor-binding domain (Ser(17)-Val(31)) of parathyroid hormone (PTH) is predicted to form an amphiphilic alpha-helix and to interact primarily with the N-terminal extracellular domain (N domain) of the PTH receptor (PTHR). We explored these hypotheses by introducing a variety of substitutions in region 17-31 of PTH-(1-31) and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Substitutions at Arg(20) reduced affinity for the intact PTHR by 200-fold or more, but altered affinity for PTHR-delNt by 4-fold or less. Similar effects were observed for Glu substitutions at Trp(23), Leu(24), and Leu(28), which together form the hydrophobic face of the predicted amphiphilic alpha-helix. Glu substitutions at Arg(25), Lys(26), and Lys(27) (which forms the hydrophilic face of the helix) caused 4-10-fold reductions in affinity for both receptors. Thus, the side chains of Arg(20), together with those composing the hydrophobic face of the ligand's putative amphiphilic alpha-helix, contribute strongly to PTHR-binding affinity by interacting specifically with the N domain of the receptor. The side chains projecting from the opposite helical face contribute weakly to binding affinity by different mechanisms, possibly involving interactions with the extracellular loop/transmembrane domain region of the receptor. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex. 相似文献
9.
Interactions between the N-terminal residues of parathyroid hormone (PTH) and the region of the PTH receptor containing the extracellular loops and transmembrane domains are thought to be critical for receptor activation. We evaluated this hypothesis by replacing the large N-terminal extracellular domain of the human type 1 PTH receptor (hP1Rc-WT) with residues 1-9 of PTH (AVSEIQLMH) using a tetraglycine linker between His-9 of the ligand and Glu-182 of the receptor near the extracellular terminus of transmembrane domain-1. Expression of this construct, hP1Rc-Tether(1-9), in COS-7 cells resulted in basal cAMP levels that were 10-fold higher than those seen in control cells transfected with hP1Rc-WT. Extending the ligand sequence to include Asn-10 and the activity-enhancing substitution of Leu-11 --> Arg yielded hP1Rc-[Arg(11)]Tether(1-11), for which we observed basal cAMP levels that were 50-fold higher than those seen with P1Rc-WT. An alanine-scan analysis of hP1Rc-[Arg(11)]Tether(1-11) revealed that Gln-6 and His-9 were not critical for autoactivation, whereas Val-2, Ile-5, and Met-8 were. The data show that tethered PTH/PTH receptors can autoactivate. Analysis of the structure-activity relationships in these tethered receptor constructs can provide new information concerning how the N-terminal residues of PTH interact with the extracellular loops and transmembrane regions of the PTH-1 receptor, particularly in regard to receptor activation. 相似文献
10.
The 10th and 11th residues of parathyroid hormone PTH(1-12) analogues were substituted to study the structure and function of PTH analogues. The substitution of Ala(10) of [Ala(3,10,12)(Leu(7)/Phe(7))Arg(11)]rPTH(1-12)NH(2) with Glu(10) and/or the Arg(11) with Ile(11) markedly decreased cAMP generating activity. Data from circular dichroism (CD) and the nuclear magnetic resonance (NMR) structural analysis of [Ala(3,10,12)(Leu(7)/Phe(7))Arg(11)]rPTH(1-12)NH(2) revealed tight alpha-helical structures, while the Glu(10) and/or Ile(11) substituted analogues showed unstable alpha-helical structures. We conclude that 10th and 11th residues are important for stabilizing its helical conformation and that destabilization of the alpha-helical structure, induced by substituting the above residues, remarkably affect its biological potency. 相似文献
11.
Jensen ML Pedersen LN Timmermann DB Schousboe A Ahring PK 《Journal of neurochemistry》2005,92(4):962-972
The present study evaluates how four key amino acid residue positions (- 4' to - 1') within the M1-M2 linker of the GABA(A) receptor beta subunit influences ion selectivity of a cation-conducting GABA receptor. Cation selectivity was found to be highly dependent on the side-chains of the amino acid residues present. The critical factor for cation selectivity was the presence of a negatively charged Glu or Asp residue in the -1' position. Receptors containing the neutral amino acids Gln or Asn or a positively charged Arg residue were anion selective. In the presence of a -1' Glu residue, the amino acids in adjacent positions were also found to be important determinants of cation selectivity. Moreover, the length of the M1-M2 linker as well as the presence of a Pro residue within this segment also affected ion selectivity, suggesting that the local environment and three-dimensional position of the -1' Glu are essential determinants of cation permeation. Conversely, no specific amino acid residues were found to be essential for anion selectivity, suggesting that the basic architecture of the selectivity segment of this class of receptor channels is optimally suited for anion conduction. 相似文献
12.
It was recently shown that the covalent tethering of the N-terminus of parathyroid hormone (PTH) to the seventh helical bundle of the G-protein coupled PTH-receptor (PTH1R) leads to autoactivation [Shimizu et al., J. Biol. Chem. 275 (2000) 19456-19460]. Here, we have developed molecular models for the interaction of PTH(1-11) tethered to PTH1R and refined them with molecular dynamics simulations. The starting structure of the ligand/receptor complex is based on experimental data from a series of spectroscopic structural studies of PTH(1-34) and the extracellular domains of PTH1R and intermolecular contact points derived from photoaffinity labeling. The resulting PTH1R/[Arg(11)]PTH(1-11) complex has the N-terminus of PTH interacting with residues of the third extracellular loop of PTH1R, as a possible mode for receptor activation. The hydrophobic residues leucine-5 and methionine-8, centrally located in the N-terminal alpha-helix of PTH(1-11), are located in deep, well-defined hydrophobic pockets in the central core of the seventh helical bundle, consistent with the requirement of these amino acids for autoactivation. We postulate that the improved signaling properties of [Arg(11)]PTH(1-11) over wild type PTH(1-11) is due to a stable hydrogen bond between Arg(11) and E444, at the beginning of TM7. The model provides atomic insight into currently available biochemical data as well as numerous putative ligand/receptor interactions, and thereby may further the rational design of reduced-size PTH agonists at the PTH1 receptor. 相似文献
13.
Gauguin L Delaine C Alvino CL McNeil KA Wallace JC Forbes BE De Meyts P 《The Journal of biological chemistry》2008,283(30):20821-20829
Current evidence supports a binding model in which the insulin molecule contains two binding surfaces, site 1 and site 2, which contact the two halves of the insulin receptor. The interaction of these two surfaces with the insulin receptor results in a high affinity cross-linking of the two receptor alpha subunits and leads to receptor activation. Evidence suggests that insulin-like growth factor-I (IGF-I) may activate the IGF-I receptor in a similar mode. So far IGF-I residues structurally corresponding to the residues of the insulin site 1 together with residues in the C-domain of IGF-I have been found to be important for binding of IGF-I to the IGF-I receptor (e.g. Phe(23), Tyr(24), Tyr(31), Arg(36), Arg(37), Val(44), Tyr(60), and Ala(62)). However, an IGF-I second binding surface similar to site 2 of insulin has not been identified yet. In this study, we have analyzed whether IGF-I residues corresponding to the six residues of the insulin site 2 have a role in high affinity binding of IGF-I to the IGF-I receptor. Six single-substituted IGF-I analogues were produced, each containing an alanine substitution in one of the following positions (corresponding insulin residues in parentheses): Glu(9) (His(B10)), Asp(12) (Glu(B13)), Phe(16) (Leu(B17)), Asp(53) (Ser(A12)), Leu(54) (Leu(A13)), and Glu(58) (Glu(A17)). In addition, two analogues with 2 and 3 combined alanine substitutions were also produced (E9A,D12A IGF-I and E9A,D12A,E58A IGF-I). The results show that introducing alanine in positions Glu(9), Asp(12), Phe(16), Leu(54), and Glu(58) results in a significant reduction in IGF-I receptor binding affinity, whereas alanine substitution at position 53 had no effect on IGF-I receptor binding. The multiple substitutions resulted in a 33-100-fold reduction in IGF-I receptor binding affinity. These data suggest that IGF-I, in addition to the C-domain, uses surfaces similar to those of insulin in contacting its cognate receptor, although the relative contribution of the side chains of homologous residues varies. 相似文献
14.
Drechsler N Fröbel J Jahreis G Gopalswamy M Balbach J Bosse-Doenecke E Rudolph R 《Biophysical chemistry》2011,154(2-3):66-72
The parathyroid hormone (PTH)1 receptor is a member of the class B G protein-coupled receptor (GPCR) family and regulates bone and mineral metabolism of vertebrates. A truncated highly active parathyroid hormone fragment PTH (1-34) exerts stimulatory effects on the receptor and is used for treatment of osteoporosis. To study the interacting amino acids of the natural peptide ligand PTH (1-84) with the ectodomain of its receptor we used peptide micro arrays on solid cellulose membranes. The amino acids Arg20 and Trp23 within the identified core binding stretch PTH (20-26) were found to be most important for affinity to the ectodomain of PTH1R. Isothermal titration calorimetry and NMR spectroscopy allowed peptide binding studies in solution and verified peptide positions required for high affinity. With this combination of biochemical and biophysical methods we extend former findings on this essential interaction and can now provide a strategy to screen for optimized therapeutic peptides. 相似文献
15.
Lysine-288 in the glucagon-like peptide-1 receptor was predicted to be ideally positioned to play a role in hormone binding. Subsequent mutation of Lys-288 to Ala or Leu greatly reduced hormone affinity, while substitution with Arg had minimal effect. Compared to wild type, the Lys288-Ala receptor had a reduced affinity for three peptide ligands with complete N-terminal sequences but not for their N-truncated analogues. Hence, the role of this positively charged residue, which is conserved at the equivalent position in all other Family B receptors, was determined to be important for receptor interaction with the N-terminal eight residues of peptide agonists. 相似文献
16.
The N-terminal domain of PTH(1-34) is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated the possibility that the side chains of residues 6 (Gln) and 10 (Gln or Asn) of PTH analogues, which would align on the same face of the predicted alpha-helix, could interact and thereby contribute to the PTH/P1R interaction process. We utilized PTH(1-11), PTH(1-14), and PTH(1-34) analogues substituted with alanine at one or both of these positions and functionally evaluated the peptides in cell lines (HKRK-B7 and HKRK-B28) stably expressing the P1R, as well as in COS-7 cells transiently expressing either the P1R or a P1R construct that lacks the amino-terminal extracellular domain (P1R-DelNt). In HKRK-B7 cells, the single substitutions of Gln(6) --> Ala and Gln(10) --> Ala reduced the cAMP-stimulating potency of [Ala(3),Gln(10),Arg(11)]rPTH(1-11)NH(2) approximately 60- and approximately 2-fold, respectively, whereas the combined Ala(6,10) substitution resulted in a approximately 2-fold gain in potency, relative to the single Ala(6) substitution. Similar effects on P1R-mediated cAMP-signaling potency and P1R-binding affinity were observed for these substitutions in [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]rPTH(1-14)NH(2). Installation of a lactam bridge between the Lys(6) and the Glu(10) side chains of [Ala(3,12),Lys(6),Glu(10),Har(11),Trp(14)]rPTH(1-14)NH(2) increased signaling potency 6-fold, relative to the nonbridged linear analogue. Alanine substitutions at positions 6 and/or 10 of [Tyr(34)]hPTH(1-34)NH(2) did not affect signaling potency nor binding affinity on the intact P1R; however, Ala(6) abolished PTH(1-34) signaling on P1R-DelNt, and this effect was reversed by Ala(10). The overall data support the hypothesis that the N-terminal portion of PTH is alpha-helical when bound to the activation domain of the PTH-1 receptor and they further suggest that intrahelical side chain interactions between residues 6 and 10 of the ligand can contribute to the receptor interaction process. 相似文献
17.
Dissecting the energetics of an antibody-antigen interface by alanine shaving and molecular grafting. 总被引:2,自引:2,他引:0
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Alanine-scanning mutagenesis on human growth hormone (hGH) identified 5 primary determinants (Arg 8, Asn 12, Arg 16, Asp 112, and Asp 116) for binding to a monoclonal antibody (MAb 3) (Jin L, Fendly BM, Wells JA, 1992, J Mol Biol 226:851-865). To further analyze the energetic importance of residues surrounding these five, we mutated all neighboring residues to alanine in groups of 7-16 (a procedure we call alanine shaving). Even the most extremely mutated variant, with 16 alanine substitutions, caused less than a 10-fold reduction in binding affinity to MAb3. By comparison, mutating any 1 of the 5 primary determinants to alanine caused a 6- to > 500-fold reduction in affinity. Replacing any of the 4 charged residues (Arg 8, Arg 16, Asp 112, and Asp 116) with a homologous residue (i.e., Arg to Lys or Asp to Glu) caused nearly as large a reduction in affinity as the corresponding alanine replacement. It was possible to graft the 5 primary binding determinants onto a nonbinding homologue of hGH, human placental lactogen (hPL), which has 86% sequence identity to hGH. The grafted hPL mutant bound 10-fold less tightly than hGH to MAb3 but bound as well as hGH when 2 additional framework mutations were introduced. Attempts to recover binding affinity by grafting the MAb3 epitope onto more distantly related scaffolds having a similar 4-helix bundle motif, such as human prolactin (23% sequence identity) or granulocyte colony-stimulating factor, were unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
Hawtin SR Wesley VJ Simms J Argent CC Latif K Wheatley M 《Molecular endocrinology (Baltimore, Md.)》2005,19(11):2871-2881
It is fundamentally important to define how agonist-receptor interaction differs from antagonist-receptor interaction. The V1a vasopressin receptor (V1aR) is a member of the neurohypophysial hormone subfamily of G protein-coupled receptors. Using alanine-scanning mutagenesis of the N-terminal juxtamembrane segment of the V1aR, we now establish that Glu54 (1.35) is critical for arginine vasopressin binding. The mutant [E54A]V1aR exhibited decreased arginine vasopressin affinity (1700-fold) and disrupted signaling, but antagonist binding was unaffected. Mutation of Glu54 had an almost identical pharmacological effect as mutation of Arg46, raising the possibility that agonist binding required a mutual interaction between Glu54 and Arg46. The role of these two charged residues was investigated by 1) substituting Glu54; 2) inserting additional Glu/Arg in transmembrane helix (TM) 1; 3) repositioning the Glu/Arg in TM1; and 4) characterizing the reciprocal mutant [R46E/E54R]V1aR. We conclude that 1) the positive/negative charges need to be precisely positioned in this N terminus/TM1 segment; and 2) Glu54 and Arg46 function independently, providing two discrete epitopes required for high-affinity agonist binding and signaling. This study explains why Glu and Arg, part of an -R(X3)L/V(X3)E(X3)L- motif, are conserved at these loci throughout this G protein-coupled receptor subfamily and provides molecular insight into key differences between agonist and antagonist binding requirements. 相似文献
19.
C Zechel D Trivedi V J Hruby 《International journal of peptide and protein research》1991,38(2):131-138
This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N-terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C-terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys17,18,Glu21]glucagon,1 a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [D-Phe4,Tyr5,Arg12]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue, 4,5,6,7-tetrahydro-1H-imidazo[c]pyridine-6-carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe6 residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip1, Lys17,18,Glu21]glucagon (2); [Tip1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (3); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (4); [dHis1,Asp3,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21+ ++]glucagon (5); des-Phe6-[Tip1,D-Phe4,Tyr5,Arg12,Glu21]glucagon (6); des-Phe6-[Asp3,D-Phe4,Tyr5,Arg12,Glu21]glucagon (7). The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3-7 are antagonists unable to activate the AC system even at concentrations as high as 10(-5) M. In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
R Kremer H P Bennett J Mitchell D Goltzman 《The Journal of biological chemistry》1982,257(23):14048-14054
Iodinated native bovine parathyroid hormone (bPTH(1-84)) was separated from uniodinated hormone by reversed-phase liquid chromatography techniques after lactoperoxidase labeling. Analysis of iodinated residues after enzymatic digestion indicated that the major labeled product was largely monoiodinated on the sole tyrosine residue. This material retained full bioactivity in an in vitro renal adenylate cyclase assay. Binding of 125I-bPTH(1-84) to rabbit renal membranes at 4 degrees C was proportional to membrane protein concentration and was saturable and dissociable. Radioligand binding was inhibited by concentrations of unlabeled bPTH(1-84) required to stimulate adenylate cyclase in the same membrane preparation but was not inhibited by non-PTH peptides other than adrenocorticotropin at high concentrations (greater than 10 microM). Synthetic NH2-terminal analogues of bPTH(1-84) all elicited approximately equivalent inhibition of radioligand binding which was, however, less potent than unlabeled bPTH(1-84), suggesting a role for the carboxyl region of the molecule in the interaction of bPTH(1-84) with its receptor. Activity of the NH2-terminal agonists was similar to bPTH(1-84) in stimulating adenylate cyclase. Although substitution in sequence position one, of serine in human PTH(1-34) for alanine in bPTH(1-34), reduced activity in the adenylate cyclase assay, inhibition of 125I-bPTH(1-84) binding by both peptides and by an analogue of bPTH(3-34) was equivalent, consistent with a minimal contribution of the first 2 residues for receptor binding of the NH2-terminal region of PTH. The results illustrate the utility of the radiolabeled preparation of native bPTH we have developed and emphasize the importance of probing the PTH receptor with an intact hormone to maximize information concerning the mechanism of PTH action. 相似文献