Identification of the Site in the Substance P (NK-1) Receptor for Modulation of Peptide Binding by Sulfhydryl Reagents

Li, Hanzhong; Hsu, Peggy P.; Sachais, Bruce S.; Krause, James E.; Leeman, Susan E.; Boyd, Norman D.

doi:10.1074/jbc.271.4.1950

Cited by 12 publications

(6 citation statements)

References 39 publications

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“…Our results have both similarities and differences from studies of the molecular basis of affinity of various gastrointestinal peptide hormone/neurotransmitters for other G-protein-coupled receptors, as well as GRP/NMB for their mammalian Bn receptors. Our results agree with the general conclusion that various peptide gastrointestinal hormone/neurotransmitter interactions with either EC’s, UTM regions or both are most important for determining the ligand’s high affinity, which is in contrast to bioactive amines, such as catecholamines, whose high affinity is determined primarily by amino acids in the TM region [25,26,40,52]. Our results showing both EC and UTM domains are important for high affinity for Univ.Lig differ from findings with a number of G-protein coupled receptor peptide ligands for other GPCRs, where only interaction with EC domains is primarily important including substance P for the NK 1 receptor [53], neuropeptide S for neuropeptide S receptor(NPS) [54], gastrin for the CCK B receptor and CCK-8 for CCK A R [55].…”

Section: Discussionsupporting

confidence: 91%

The molecular basis for high affinity of a universal ligand for human bombesin receptor (BnR) family members

Uehara

Hocart²,

González

et al. 2012

Biochemical Pharmacology

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There is increased interest in the Bn-receptor family because they are frequently over/ectopically-expressed by tumors and thus useful as targets for imaging or receptor-targeted-cytotoxicity. The synthetic Bn-analog,[D-Tyr6,β-Ala11,Phe13,Nle14]Bn(6-14)[Univ.Lig] has the unique property of having high affinity for all three human BNRs(GRPR,NMBR,BRS-3), and thus could be especially useful for this approach. However, the molecular basis of this property is unclear and is the subject of this study. To accomplish this, site-directed mutagenesis was used after identifying potentially important amino acids using sequence homology analysis of all BnRs with high affinity for Univ.Lig compared to the Cholecystokinin-receptor(CCKAR), which has low affinity. Using various criteria 74 amino acids were identified and 101 mutations made in GRPR by changing each to those of CCKAR or to alanine. 22 GRPR mutations showed a significant decrease in affinity for Univ.Lig(>2-fold) with 2 in EC2[ D97N,G112V], 1 in UTM6[Y284A], 2 in EC4[R287N,H300S] showing >10-fold decrease in Univ.Lig affinity. Additional mutations were made to explore the molecular basis for these changes. Our results show that high affinity for Univ.Lig by human Bn-receptors requires positively charged amino acids in extracellular (EC)-domain 4 and to a lesser extent EC2 and EC3 suggesting charge-charge interactions may be particularly important for determining the general high affinity of this ligand. Furthermore, transmembrane amino acids particularly in UTM6 are important contributing both charge-charge interactions as well as interaction with a tyrosine residue in close proximity suggesting possible receptor-peptide cation-pi or H–bonding interactions are also important for determining its high affinity.

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Section: Discussionsupporting

confidence: 91%

The molecular basis for high affinity of a universal ligand for human bombesin receptor (BnR) family members

Uehara

Hocart²,

González

et al. 2012

Biochemical Pharmacology

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“…Compared with the NMBR and GRPR, our results are similar in that for selectivity of NMB or GRP, with both extracellular and transmembrane regions being important (Fathi et al, 1993a;Akeson et al, 1997;Tokita et al, 2001b;Nakagawa et al, 2005). Compared with other GPCRs, our results are similar to the peptide agonist, substance P with NK1 receptors (Li et al, 1996), and CCK-8 with CCK-B receptors (Silvente-Poirot et al, 1998), where both the extracellular and transmembrane domains are important for selectivity. In contrast, the affinity of CCK-8 for CCK-A receptor (Silvente-Poirot et al, 1998) or secretin for secretin receptors (Holtmann et al, 1995) is determined by the extracellular domains, whereas differences in the transmembrane regions play the critical role in the affinity of nociceptin for orphanin FQ receptors (Meng et al, 1996).…”

Section: Discussionsupporting

confidence: 75%

Molecular Basis for Agonist Selectivity and Activation of the Orphan Bombesin Receptor Subtype 3 Receptor

González¹,

Hocart²,

Portal‐Núñez³

et al. 2007

J Pharmacol Exp Ther

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Bombesin receptor subtype (BRS)-3, a G-protein-coupled orphan receptor, shares 51% identity with the mammalian bombesin (Bn) receptor for gastrin-releasing peptide. There is increasing interest in BRS-3 because it is important in energy metabolism, glucose control, motility, and tumor growth. BRS-3 has low affinity for all Bn-related peptides; however, recently synthetic high-affinity agonists, [D-Tyr 6 /D-Phe 6 ,␤Ala 11 ,Phe 13 ,Nle 14 ]Bn-(6 -14), were described, but they are nonselective for BRS-3 over other Bn receptors. Based on these peptides, three BRS-3-selective ligands were developed: peptide 2, [D-Tyr ]Bn(6 -14); and peptide 4, acetyl-Phe-Trp-Ala-His-(tBzl)-piperidine-3 carboxylic acid-Gly-Arg-NH 2 . Their molecular determinants of selectivity/high affinity for BRS-3 are unknown. To address this, we used a chimeric/site mutagenesis approach. Substitution of extracellular domain 2 (EC2) of BRS-3 by the comparable gastrin-releasing peptide receptor (GRPR) domain decreased 26-, 4-, and 0-fold affinity for peptides 4, 3, and 2. Substitution of EC3 decreased affinity 4-, 11-, and 0-fold affinity for peptides 2 to 4. Ten-point mutations in the EC2 and adjacent transmembrane regions (TM2) 2 and 3 of BRS-3 were made. His107 (EC2-BRS-3) for lysine (H107K) (EC2-GRPR) decreased affinity (25-and 0-fold) for peptides 4 and 1; however, it could not be activated by either peptide. Its combination with Val101 (TM2), Gly112 (EC2), and Arg127 (TM3) resulted in complete loss-ofaffinity of peptide 4. Receptor-modeling showed that each of these residues face inward and are within 4 Å of the binding pocket. These results demonstrate that Val101, His107, Gly112, and Arg127 in the EC2/adjacent upper TMs of BRS-3 are critical for the high BRS3 selectivity of peptide 4. His107 in EC2 is essential for BRS-3 activation, suggesting amino-aromatic ligand/ receptor interactions with peptide 4 are critical for both binding and activation. Furthermore, these result demonstrate that even though these three BRS-3-selective agonists were developed from the same template peptide,

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“…Compared with GRPR/BRS-3, our results are similar in that both extracellular and upper TM regions are important for selectivity of Bn, GRP, and some BRS-3-selective peptide agonists for GPRR or BRS-3 Tokita et al, 2002;Nakagawa et al, 2005;Gonzalez et al, 2008;Jensen et al, 2008). Compared with other G-protein-coupled receptors, our results are similar to the interaction of the peptide agonists, substance P with NK-1 receptors (Li et al, 1996) and CCK-8 with CCK-B receptors (Silvente-Poirot et al, 1998), where both the extracellular and TM domains play a marked role in selectivity. However, the affinity of CCK-8 for the CCK-A receptor (Silvente-Poirot et al, 1998), secretin for secretin receptors (Holtmann et al, 1995), or the GRPR peptide antagonist JMV641 (Tokita et al, 2001b) for GRPR is determined by extracellular domains, whereas differences in only TM regions are crucial for affinity of nociceptin for orphanin-FQ receptors (Meng et al, 1996) or the peptoid antagonist PD168368 for NMBR (Tokita et al, 2001a).…”

Section: Discussionsupporting

confidence: 71%

Molecular Basis for the Selectivity of the Mammalian Bombesin Peptide, Neuromedin B, for Its Receptor

González

Nakagawa²,

Mantey³

et al. 2009

J Pharmacol Exp Ther

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The mammalian bombesin (Bn) peptides, neuromedin B (NMB) and gastrin-releasing peptide (GRP), have widespread actions in many tissues, and their effects are mediated by two closely related G-protein-coupled receptors, the NMBR and GRPR. Little is known about the structural determinants of NMBR selectivity for NMB, in contrast to GRP selectivity for the GRPR, which has been extensively studied. To provide insight, chimeric NMBR-GRPR loss-of-affinity and gain-of-affinity mutants were made, as well as NH 2 -terminally truncated NMBR and point mutants using site-directed mutagenesis. Receptors were expressed in Balb-3T3-cells or CHOP cells, and affinities were determined. NMB had 115-fold greater affinity for NMBR than GRPR. Receptor-chimeric studies showed that NMBR selectivity for NMB was primarily determined by differences in the third extracellular (EC3) regions of GRPR-NMBR and adjacent upper-transmembrane-5 (TM5) region. In this region, 24 NMB gain-of-affinity GRPR mutants or NMBR loss-of-affinity point/ combination mutants were made. Three gain-of-affinity mutant GRPRs [[A198I] (EC3), [H202Q] (EC3), [S215I] (upper TM5)]had increased NMB affinity (2.4 -21-fold), and these results were confirmed with NMBR loss-of-affinity mutants [I199A, Q203H,I215S-NMBR]. The combination mutant [A198I, S215]GRPR had the greatest effect causing a complete NMB gain-of-affinity. The importance of differences at position 199NMBR or 203NMBR was studied by substituting amino acids with various properties. Our results show that NMBR selectivity for NMB is due to differences in the EC3 of NMBR-GRPR and the adjacent upper-TM5 region. Within these regions, isoleucines in NMBR [position 199 (EC3)] (instead of A198GRPR) and in 215NMBR (TM5) (instead of S214GRPR), as well as Q203NMBR (instead of H202GRPR) are responsible for high NMB-affinity/selectivity of NMBR. The effect at position 199 is primarily due to differences in hydrophobicity of the substitution, whereas steric factors and charge of the substitution at position 203 were important determinants of NMB selectivity.

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Identification of the Site in the Substance P (NK-1) Receptor for Modulation of Peptide Binding by Sulfhydryl Reagents

Cited by 12 publications

References 39 publications

The molecular basis for high affinity of a universal ligand for human bombesin receptor (BnR) family members

The molecular basis for high affinity of a universal ligand for human bombesin receptor (BnR) family members

Molecular Basis for Agonist Selectivity and Activation of the Orphan Bombesin Receptor Subtype 3 Receptor

Molecular Basis for the Selectivity of the Mammalian Bombesin Peptide, Neuromedin B, for Its Receptor

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