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T M Fong

Publications and source records attributed to T M Fong.

At least 37 records · Page 2Linked to original sources
PubMed1995

Molecular characterization of a common binding site for small molecules within the transmembrane domain of G-protein coupled receptors.

The cloning of over 100 members of the superfamily of G-protein coupled receptors has resulted in the identification and characterization of novel targets for therapeutic intervention. In addition, mutagenesis studies aimed at understanding the nature of the molecular interactions of these receptors with peptides and small molecules have led to advancements in the ability to synthesize novel therapeutic agents with high affinity and specificity for these receptors. These experiments have shown that there is a common binding site for small molecules within the transmembrane domain of G-protein coupled receptors regardless of the nature of the endogenous, physiologically relevant receptor agonist. Finally, the demonstration that the binding sites for peptide agonists and nonpeptide competitive antagonists are not necessarily identical has provided insights regarding the mechanism of competitive antagonism in these receptors.

Amino Acid Sequence↗
PubMed1995

Mutational analysis of neurokinin receptor function.

The interactions of the NK1 receptor with peptide agonists or nonpeptide antagonists have been investigated by site-directed mutagenesis and computer modeling. At least 10 residues in the extracellular and transmembrane regions of the receptor are required for the binding of many peptide agonists. The C-terminal amide of peptide agonists is likely to be bound near Asn-85. Residues likely to be involved in the subsequent receptor activation include Glu-78 and Tyr-205. The binding site for nonpeptide antagonists can be defined by at least five residues in transmembrane helices 4-7, and primary contacts between key residues and quinuclidine antagonists have been assigned based on CP-96,345 and its analogs. Analyses of the wild-type and mutant NK1 and NK2 receptors, intact and truncated peptides, and various antagonists suggest that the agonist and antagonist binding sites overlap spatially, even though agonists and antagonists do not interact with the same set of residues on the receptor. Mapping the ligand binding site not only allows us to better understand the ligand--receptor interaction and antagonism but also leads to a refined three-dimensional model of the NK1 receptor.

Amino Acid Sequence↗
PubMed1995

The family of G-protein-coupled receptors.

The family of G-protein-coupled receptors can be defined by their similar structural and functional characteristics. Although their primary sequences are quite diverse, these proteins share several common structural features that reflect their common mechanism of action. Mutagenesis and biophysical analysis of several of these receptors indicate that small molecule agonists and antagonists bind to a hydrophobic pocket buried in the transmembrane core of the receptor. In contrast, peptide ligands bind to both the extracellular and transmembrane domains. The mechanisms by which these peptide and small molecule agonists cause receptor activation are being explored by various approaches, but are not yet well defined. A deeper understanding of structural basis for the functional activity of this large family of receptors will have important implications for drug design in a variety of therapeutic areas.

Binding Sites↗
PubMed1994

Characterization of a fluorescent substance P analog.

We describe the development and characterization of substance P labeled at Lys3 with fluorescein ([fluorescein Lys3]SP) as a fluorescent probe for the neurokinin 1 (NK1) receptor. [fluorescein Lys3]SP is an agonist at the human NK1 receptor, with an affinity for both the high-affinity and low-affinity binding states of the receptor approximately 6-fold lower than that of substance P. Binding of the probe to the human NK1 receptor expressed in Sf9 insect cells was observed directly by monitoring either a decrease in fluorescence intensity or an increase in anisotropy of the [fluorescein Lys3]SP. Detection by anisotropy gave the larger signal and thus was used to characterize the interaction of [fluorescein Lys3]SP with the receptor. The anisotropy of the bound ligand was 0.17, compared to 0.04 for the free ligand. The fluorescence was quenched by about 15% upon binding to the receptor. Bound [fluorescein Lys3]SP was displaced by unlabeled SP and by the quinuclidine antagonist L-703,606. As expected for an agonist, binding was also reduced by the addition of the nonhydrolyzable guanine nucleotide analog GppNHp. [fluorescein Lys3]SP should provide a useful structural and kinetic probe for the NK1 receptor.

Amino Acid Sequence↗
PubMed1994

Interaction of glutamine 165 in the fourth transmembrane segment of the human neurokinin-1 receptor with quinuclidine antagonists.

Substance P binds to and activates the neurokinin-1 receptor with high affinity, thereby modulating several neuronal pathways including pain transmission and neurogenic inflammation. Several high affinity non-peptide antagonists have recently been described. To elucidate the molecular interactions specific for binding to the neurokinin-1 receptor, site-directed mutagenesis has been utilized to identify amino acid residues that interact directly with antagonists. Glutamine 165 in the fourth transmembrane segment was shown to be critical for the binding of CP-96,345 but not SR140333. Analysis of quinuclidine analogs suggests that glutamine 165 interacts with the C-3 heteroatom in this class of antagonists, probably through a hydrogen bond. Glutamine 165 also plays a minor role in the binding of peptides and RP67580. In contrast, serine 169 was determined to be critical for the binding of RP67580. These data indicate that residues 165 and 169 in the fourth transmembrane segment, along with residues in the fifth, sixth, and seventh transmembrane segments as demonstrated previously, form the non-peptide antagonist binding site in the neurokinin-1 receptor. Furthermore, the antagonist binding site overlaps with the binding site for peptide agonists in the fourth and seventh transmembrane segments.

Amino Acid Sequence↗
PubMed1994

Interaction of substance P with the second and seventh transmembrane domains of the neurokinin-1 receptor.

The neurokinin-1 receptor is a member of the G-protein-coupled receptor family and has the highest affinity for the endogenous peptide transmitter substance P. Previous studies have indicated that several residues in the first and second extracellular segments, and at least part of the transmembrane domain, of the human neurokinin-1 receptor are involved in substance P binding to the receptor. To further map the peptide binding site, single-residue substitutions in the transmembrane domains were analyzed. Asn-85, Asn-89, Tyr-92, and Asn-96 in the second transmembrane domain and Tyr-287 in the seventh transmembrane domain are required for the high-affinity binding of peptides, with Asn-85 possibly interacting with the C-terminus of substance P. In addition, Glu-78 in the second transmembrane domain and Tyr-205 in the fifth transmembrane domain appear to be involved in the receptor activation process. Some of the key residues for peptide binding are likely to be near those residues that are required for the binding of competitive antagonists (such as His-197, His-265, and Tyr-287). These data suggest that a volume exclusion effect can explain the competitive antagonism of substance P binding by non-peptide antagonists. Furthermore, the key residues identified thus far are required for the high-affinity binding of all three neurokinin peptides, consistent with a hypothesis that the conformational compatibility between the receptor and the peptide agonist may be a major determinant of peptide recognition.

Amino Acid Sequence↗
PubMed1994

The role of histidine 265 in antagonist binding to the neurokinin-1 receptor.

Previous studies suggested that the antagonist binding site in the neurokinin-1 receptor is composed of phylogenetically conserved residues, while phylogenetically divergent residues affect the conformation of the binding site. To test this hypothesis, we investigated the role of conserved residues in antagonist binding. Histidine 197 in the human receptor was found to interact with CP-96,345 but not RP67580. In addition, a nearby residue, histidine 265 of the human receptor, is required for the binding of RP67580 but not CP-96,345 or substance P. The interaction between residue 265 and RP67580 is consistent with a hydrogen bonding interaction. Analysis of several analogs of CP-96,345 revealed that histidine 265 of the human receptor is in proximity to the substituted benzyl moiety of CP-96,345, and can interact with other analogs of CP-96,345. In contrast to the human neurokinin-1 receptor, both histidine 197 and histidine 265 in the rat neurokinin-1 receptor appear to interact with both CP-96,345 and RP67580. These results support a conformational difference between the antagonist binding sites of the rat and human neurokinin-1 receptors and provide a model for examining specific interactions between antagonists and the receptor.

Amino Acid Sequence↗
PubMed1994

Expression and solubilization of a recombinant human neurokinin-1 receptor in insect cells.

The human neurokinin-1 receptor has been expressed in insect cells using a recombinant baculovirus. The expression level is about 10 times higher than that obtained in mammalian cells. The recombinant receptor was solubilized with CHAPS, and a PEG precipitation procedure was shown to be effective in regaining high affinity substance P binding. This system should allow large scale purification of the human neurokinin-1 receptor.

Animals↗
PubMed1994

Localization of the ligand binding site of the neurokinin-1 receptor: interpretation of chimeric mutations and single-residue substitutions.

Previous studies have indicated that substitution of the third or fourth extracellular segment of the human neurokinin-1 receptor with the equivalent segment from the neurokinin-3 receptor affects the binding affinities of peptide agonists and/or nonpeptide antagonists. To elucidate the roles of the divergent residues within these domains in ligand binding, single-residue substitutions in these regions were analyzed. Neurokinin B affinity was increased by one single-residue substitution (E172K) in the third extracellular segment and one double-residue replacement (L279R+K280W) in the fourth extracellular segment, and the affinity for the antagonist L-703,606 was reduced by one single-residue substitution (Y272A) in the fourth extracellular segment. The effect of these three specific substitutions is consistent with the prediction of chimeric mutations. However, the substantial reduction in ligand binding affinity observed upon multiple-residue substitutions in the third extracellular segment (residues 176-183 or 187-195) has not been reproduced by eliminating potential electrostatic interactions or substituting with the corresponding residues from the neurokinin-3 receptor, suggesting that the reduction in ligand binding affinity observed with some chimeric receptors is not due to the loss of direct electrostatic interactions. These data indicate that other factors such as conformational effects may complicate the interpretation of data obtained with chimeric receptors, and they demonstrate the need to evaluate chimeric receptors along with single-residue substitutions in the same region to localize specific residues involved in ligand binding. Furthermore, the available data suggest that one major determinant of peptide selectivity in the neurokinin-1 receptor may be the conformational compatibility between a peptide and the receptor.

Amino Acid Sequence↗
PubMed1993

Thapsigargin blocks the mobilisation of intracellular calcium caused by activation of human NK1 (long) receptors expressed in Chinese hamster ovary cells.

Neurokinin receptors can couple to several second messenger systems including phospholipase C and intracellular calcium mobilisation. Using FURA-2 microspectrofluorimetry, this study examines the mobilisation of calcium in CHO cells which had been stably transfected with the long isoform of the human NK1 receptor. Substance P caused a concentration-dependent rise in inositol phosphate production which was correlated with a reversible increase in intracellular calcium levels. The mobilisation of calcium was reduced by the removal of extracellular calcium from the bathing solution, and almost totally abolished by depletion of intracellular calcium pools with 1 microM thapsigargin. These data suggest that the transduction mechanism associated with NK1 (long) receptor activation can utilise both intracellular and extracellular calcium pools in this expression system.

Animals↗
PubMed1993

Amino-aromatic interaction between histidine 197 of the neurokinin-1 receptor and CP 96345.

Substance P is a peptide neurotransmitter that binds to the neurokinin-1 receptor and is involved in pain transmission and neurogenic inflammation. Recently, a non-peptide substance P antagonist (CP 96345) has been shown to be effective in animal models of pain and inflammation. An understanding of the molecular interactions responsible for ligand binding will be critical for the development of more specific and selective antagonists for the neurokinin-1 receptor and for the discovery of new antagonists for related G-protein-coupled receptors. Here we report that histidine at position 197 in the fifth transmembrane helix of the human neurokinin-1 receptor binds specifically to CP 96345 but not to peptide agonists. Substitution of His 197 by different amino acids and analysis of structural analogues of antagonists suggest that His 197 is involved in an amino-aromatic interaction with the benzhydryl moiety of CP 96345.

Amino Acid Sequence↗
PubMed1993

Characterization of the binding of [125I-iodo-histidyl, methyl-Phe7] neurokinin B to the neurokinin-3 receptor.

We have characterized the binding of [125I-iodo-histidyl, methyl Phe7]neurokinin B (125I-NKB) to the human neurokinin-3 (NK3) receptor. 125I-NKB specifically binds to the NK3 receptor expressed in CHO cells with a Kd of 0.2 nM. The ligand displays little crossreactivity with the human NK1 and NK2 receptors. The binding of 125I-NKB to the human NK3 receptor and to rat cortex membranes is inhibited by neurokinin B with IC50 of 1.5 nM and 4 nM, respectively. In contrast, 350- to 500-fold higher concentrations of substance P and neurokinin A are required to inhibit binding to either receptor preparation. The data suggest that 125I-NKB is a high affinity, selective ligand for the human and rat NK3 receptor.

Animals↗
PubMed1992

Localization of agonist and antagonist binding domains of the human neurokinin-1 receptor.

To identify the molecular determinants of ligand-receptor interactions, the extracellular domain of the human neurokinin-1 receptor was systematically substituted with the corresponding sequences from the other two neurokinin receptor subtypes. Three residues within the first extracellular segment and 2 residues of the second segment are required for the optimal binding of all three natural peptide agonists. The divergent nature of 4 of the 5 residues supports the hypothesis that the peptide binding site on the neurokinin-1 receptor is not highly conserved in the other two receptor subtypes. In contrast, substitution of part of the third extracellular segment and the fourth extracellular segment with the corresponding amino acids of the human neurokinin-3 receptor results in an increase in neurokinin B affinity without affecting substance P binding, suggesting that the two peptides do not interact with the same set of functional groups on the receptor. Among the four extracellular regions, only parts of the third and fourth segments affect the binding of the quinuclidine antagonist L-703,606, and these two regions may partially account for the neurokinin-1 receptor subtype specificity of this non-peptide antagonist. These studies demonstrate that both the extracellular and transmembrane domains of the neurokinin-1 receptor are involved in the binding of substance P and related peptides.

Amino Acid Sequence↗
PubMed1992

Molecular basis for the species selectivity of the neurokinin-1 receptor antagonists CP-96,345 and RP67580.

Two non-peptide substance P antagonists exhibit opposite rank orders of potency for the human and rat neurokinin-1 receptors. CP-96,345 shows selectivity for the human receptor, whereas RP67580 shows selectivity for the rat receptor. Amino acid sequence comparison of the two receptors reveals 22 divergent residues. To elucidate the molecular basis for the species selectivity of these antagonists, divergent residues in the human neurokinin-1 receptor were substituted by the rat homologs. Analysis of mutant receptors revealed that substitution of 2 residues (V116L and I290S) in the transmembrane domain of the human neurokinin-1 receptor is both necessary and sufficient to reproduce the antagonist binding affinities of the rat receptor. The nature of these substitutions and the magnitude of the changes in binding affinity suggest that residues 116 and 290 do not interact directly with the antagonist molecules. The present results support a model in which phylogenetically conserved residues interact directly with the antagonists, while phylogenetically divergent residues affect the local helical packing of the receptor. Such a change in local structure would lead to increased binding affinity for one class of antagonists and decreased affinity for another.

Amino Acid Sequence↗
PubMed1992

The extracellular domain of the neurokinin-1 receptor is required for high-affinity binding of peptides.

The neurokinin-1 receptor binds neurokinin peptides with the potency order of substance P > substance K > neurokinin B. Elucidating the molecular basis of differential peptide selectivity will require the localization of the binding domain on the receptor. In the present report, mutagenesis and heterologous expression experiments reveal that a segment of the extracellular N-terminal sequence of the neurokinin-1 receptor is required for the high-affinity binding of substance P and related peptide agonists. Substitution of amino acid residues in the N-terminal region of the receptor affects the binding affinity of both intact peptides and a C-terminal substance P "analog", but not of a nonpeptide antagonist. Glycosylation of the receptor does not change the peptide binding affinity. In addition, substitution of the valine-97 residue in the rat neurokinin-1 receptor by a glutamate residue increases the binding affinity of neurokinin B but not substance P or substance K, suggesting that the second extracellular segment is involved in peptide selectivity. These results indicate that the extracellular domains of neurokinin-1 receptor play a critical role in peptide binding.

Amino Acid Sequence↗