Biomedical subjects

C D Strader

Publications and source records attributed to C D Strader.

At least 55 records · Page 3Linked to original sources

PubMed1995

Conformational compatibility as a basis of differential affinities of tachykinins for the neurokinin-1 receptor.

The neurokinin-1 receptor is characterized by sub-nanomolar affinity for substance P and 30-100 nM affinity for other substance P-related peptides, including neurokinin B and septide. We have characterized a neurokinin-1 receptor mutant containing a Y216A substitution in the fifth transmembrane segment. This receptor mutant binds substances P with sub-nanomolar affinity but loses much of its peptide discrimination capability, exhibiting 1-2 nM binding affinity for other tachykinins. Kinetic measurements of ligand binding indicate that the increased binding affinity of neurokinin B and septide for the Y216A mutant compared to the wild-type receptor is due to a 100-fold increase in the association rate constant without appreciable change in the dissociation rate constant. The substantially increased association rate constant for the Y216A mutant suggests that the mutant receptor is probably more flexible in accommodating the approaching peptide molecule. It is proposed that a major determinant of peptide specificity for the wild-type neurokinin-1 receptor is the overall conformational compatibility between the receptor and the ligand, rather than residue-specific interactions with the divergent N-terminal residues of different peptides. Furthermore, the highly conserved nature of Tyr-216 in the G protein coupled receptor family suggests that this residue may also play an important role in the receptor activation process in general.

Amino Acid Sequence↗

PubMed1995

Interaction of [fluorescein-Trp25]glucagon with the human glucagon receptor expressed in Drosophila Schneider 2 cells.

The human glucagon receptor was expressed at high density in Drosophila Schneider 2 (S2) cells. Following selection with G418 and induction with CuSO4, the cells expressed the receptor at a level of 250 pmol/mg of membrane protein. The glucagon receptor was functionally coupled to increases in cyclic AMP in S2 cells. Protein immunoblotting with anti-peptide antibodies revealed the expressed receptor to have an apparent molecular mass of 48 kDa, consistent with low levels of glycosylation in this insect cell system. Binding of [fluorescein-Trp25]glucagon to S2 cells expressing the glucagon receptor was monitored as an increase in fluorescence anisotropy along with an increase in fluorescence intensity. Anisotropy data suggest that the mobility of the fluorescein is restricted when the ligand is bound to the receptor. Kinetic analysis indicates that the binding of glucagon to its receptor proceeds via a bimolecular interaction, with a forward rate constant that is several orders of magnitude slower than diffusion-controlled. These data would be consistent with a conformational change upon the binding of agonist to the receptor. The combination of [fluorescein-Trp25]glucagon with the S2 cell expression system should be useful for analyzing glucagon receptor structure and function.

Amino Acid Sequence↗

PubMed1995

Identification of residues involved in ligand binding to the neurokinin-2 receptor.

Several residues of the human neurokinin-2 receptor have been identified to be critical for the binding of peptide agonists and non-peptide antagonists. Amino acid substitutions in the first and second extracellular segments and the second transmembrane segment led to substantial reduction in peptide affinity without affecting the affinity of antagonist SR48968. These effects are identical to those observed for homologous residues in the neurokinin-1 receptor, suggesting that these three regions are involved in high-affinity peptide binding to both receptor subtypes. On the other hand, some conserved residues in the fourth to seventh transmembrane segments are required for peptide binding to only one receptor subtype but not both. The conserved nature and location of these receptor residues suggest that the distance between bound peptide and helices 4-7 varies depending on the receptor subtype. It is likely that the conformational compatibility between a ligand and a given receptor determines the magnitude of binding affinity, and thus receptor subtype selectivity. While many single-residue substitutions did not affect the binding affinity of the antagonist SR48968, two double mutants in the sixth and seventh transmembrane segments were found to reduce its affinity substantially. Therefore, receptor residues participate cooperatively in the binding of SR48968. These results demonstrate the usefulness of combining single-residue substitutions in studying and confirming the role of receptor residues in ligand binding. Finally, the overlapping nature of agonist and antagonist binding sites is consistent with the observation that substitutions of some residues modify the binding affinities of both peptide agonists and non-peptide antagonists.

Amino Acid Sequence↗

PubMed1995

Targeted disruption of a B2 bradykinin receptor gene in mice eliminates bradykinin action in smooth muscle and neurons.

Mice that are homozygous for the targeted disruption of the gene encoding the B2 bradykinin receptor have been generated. The gene disruption results in a deletion of the entire coding sequence for the B2 receptor. The disruption of the B2 receptor gene has been confirmed by genetic, biochemical, and pharmacological analyses. Mice that are homozygous for the disruption of the B2 receptor gene are fertile and indistinguishable from their littermates by visual inspection. Bradykinin fails to produce responses in pharmacological preparations from ileum, uterus, and the superior cervical ganglia from these mice. Therefore, expression of a single gene appears to be responsible for conferring responsiveness to bradykinin in these tissues.

Animals↗

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

Isolation of a preadipocyte cell line from rat bone marrow and differentiation to adipocytes.

A unique population of rat adipocyte precursor cells was derived from normal rat bone marrow. The epitheloid-like preadipocytes were isolated from a mixed culture of bone marrow cells by a combination of differential trypsinization, enrichment by Ficoll gradient centrifugation, and differential seeding. This cell line, designated RBM-Ad, can be fully differentiated into multilocular adipocytes morphologically resembling brown adipose tissue. No changes in the differentiation pattern are observed during propagation of these cells, and they have been successfully carried and differentiated up to passage 49. Histological staining of differentiated cells with Sudan black, Sudan IV, and oil red O indicates the presence of lipids in intracellular vesicles. The nonselective beta-adrenergic agonist isoproterenol stimulates adenylyl cyclase activity in both preadipocytes and differentiated adipocytes. In contrast, BRL-37344, a beta 3-adrenergic receptor-specific agonist, stimulates adenylyl cyclase activity and glycerol release in differentiated adipocytes, but not preadipocytes. In addition, differentiated adipocytes contain messenger RNA encoding the brown adipose-specific protein, thermogenin. Thus, this rat preadipocyte cell line can be differentiated into adipocytes that histologically and functionally resemble brown adipose tissue.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Characterization of the interaction of diacylpiperazine antagonists with the human neurokinin-1 receptor: identification of a common binding site for structurally dissimilar antagonists.

We recently described a novel series of diacylpiperazine antagonists of the human neurokinin (NK)-1 receptor. The diacylpiperazine compounds are structurally dissimilar from previously described NK-1 antagonists. L-161,664 [1-(N,N-diphenylaminocarbonyl)-4-(N',N'-di-n-pentylaminocarbony l) piperazine-2-diethylaminopropylcarboxamide] inhibits 125I-substance P binding to the human NK-1 receptor with an IC50 of 43 +/- 21 nM but has 50-fold and 200-fold lower affinity for the human NK-2 and NK-3 receptors, respectively. L-161,664 inhibits substance P-stimulated inositol monophosphate accumulation in Chinese hamster ovary cells expressing the human NK-1 receptor by increasing the EC50 for substance P but not its maximal effect. The compound decreases the apparent affinity of the NK-1 receptor for 125I-substance P and does not alter the rate of dissociation of 125I-substance P from the receptor. These data indicate that L-161,664 is a potent and selective competitive antagonist of the human NK-1 receptor. L-161,664 has reduced affinity for mutants of the NK-1 receptor in which alanine has replaced Gln-165 in transmembrane helix 4, His-197 in helix 5, His-265 in helix 6, or Tyr-287 in helix 7. Similarly, a novel series of acyclic 2-benzhydryl-2-aminoethyl ethers that we have recently shown to be competitive NK-1 receptor antagonists have reduced affinity for the Q165A. H197A, and H265A mutant receptors. These residues have been shown to be important for binding of quinuclidine, tryptophan benzyl ester, and perhydroisoindole antagonists to the receptor. Analysis of the interaction of structural analogs of L-161,664 with the Q165A mutant receptor suggests that this residue interacts with the 2-diethylaminopropylcarboxamide side chain of L-161,664. Thus, even though the diacylpiperazine antagonists are structurally dissimilar from other classes of antagonists described to date, these data suggest that a common antagonist binding site that accomodates much structural diversity is present in the human NK-1 receptor. Furthermore, these data, combined with those obtained from medicinal chemistry approaches, suggest a minimum pharmacophore map for the interaction of these diverse ligands with the NK-1 binding site.

Amino Acid Sequence↗

PubMed1995

Determination of structural domains for G protein coupling and ligand binding in beta 3-adrenergic receptor.

The beta 3-adrenergic receptor (beta 3AR) is a member of the super-family of G protein-coupled receptors that are characterized by seven putative transmembrane helices connected by hydrophilic loops. The mechanism by which the activated beta ARs transmit the signals across the plasma membrane involves the stimulation of Gs, which in turn activates adenylyl cyclase, yielding the second messenger cAMP. In the present study, we created a series of mutant beta 3ARs to explore the structural basis for the subtype-specific binding of BRL 37344, a beta 3-selective agonist, and for the coupling of the receptor to Gs. To study the mechanism of subtype-specific binding of BRL 37344, chimeric beta 2/beta 3ARs were constructed and expressed in Raji cells. Binding studies suggest that the transmembrane segment 5 region of the beta 3AR contains critical determinants for observed high affinity for BRL 37344. Previous studies of beta 2ARs have demonstrated a role for the third intracellular loop in activating Gs. To investigate the role of this region in the beta 3AR, we constructed mutant beta 3ARs lacking a small segment of the amino- or carboxyl-terminal domain of the third intracellular loop. Expression of these mutant receptors in mouse L cells and Raji cells reveals that although both mutants are capable of binding the antagonist [125l]iodocyanopindolol, the agonist-stimulated cAMP production mediated by these mutant receptors is markedly attenuated or abolished. In addition, both mutant beta 3ARs exhibit an approximately 10-fold increase in affinity for agonist binding, whereas the affinity for antagonists is not affected. This increased agonist affinity is not altered by treatment with 100 microM 5' quanylyl-imidodiphosphate, suggesting that these mutant receptors are uncoupled from G proteins. The results of the present study demonstrate that these regions of the third intracellular loop of beta 3AR are critical for coupling to G proteins and suggest a role for these regions in maintaining the resting state of the unliganded receptor.

Adrenergic beta-Agonists↗

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↗

PubMed1995

Tissue-specific pharmacology of testosterone and 5 alpha-dihydrotestosterone analogues: characterization of a novel canine liver androgen-binding protein.

The mechanism by which the hormones 5 alpha-dihydrotestosterone and testosterone differentially regulate such diverse functions as development of male internal and external genitalia and maintenance of prostatic growth via a single androgen receptor (AR) is not well understood. To search for potential AR isoforms, an extensive pharmacological survey of the binding of [3H]mibolerone (7 alpha,17 alpha-[3H]dimethyl-19-nortestosterone) in dog prostate, adrenal gland, testis, liver, kidney, brain, muscle, and spleen cytosolic extracts was carried out. The antagonist androst-4-en-3,17-dione (ATD), as well as a series of unsaturated analogues of testosterone, exhibited marked tissue specificity for binding to mibolerone-binding proteins (MBPs), with ATD having a 10-fold higher affinity for the MBPs present in liver than for those in prostate and testis. The difference in affinity was not due to tissue-specific metabolism of ATD. Competition binding profiles for ATD with mixtures of prostate and liver extracts were consistent with two distinct populations of binding sites. Both wild-type human AR-B and the recently discovered human AR-A isoform were expressed in COS cells and were found to exhibit pharmacology similar to that of the prostatic MBPs in dogs. Analogues of ATD or testosterone could prove to be useful probes for delineating the differential effects of 5 alpha-dihydrotestosterone and testosterone on the biological actions of the AR and related proteins.

Androgen-Binding Protein↗

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

Expression cloning of a human B1 bradykinin receptor.

A cDNA clone encoding a human B1 bradykinin receptor was isolated from a human embryonic lung fibroblast cDNA library by expression cloning. The photoprotein aequorin was utilized as an indicator of the ability of the B1 receptor agonist [des-Arg10]kallidin to mediate Ca2+ mobilization in Xenopus laevis oocytes injected with RNA. A clone was isolated with a 1307-nucleotide insert which contains an open reading frame encoding a 353-amino acid protein with the characteristics of a G-protein-coupled receptor. The amino acid sequence of the B1 bradykinin receptor is 36% identical to the amino acid sequence of the B2 bradykinin receptor. The cloned B1 bradykinin receptor expressed in mammalian cells exhibits high affinity binding for 3H-labeled [des-Arg10]kallidin and low affinity for bradykinin. The B1 receptor antagonist [des-Arg10,Leu9]kallidin effectively displaces 3H-labeled [des-Arg10]kallidin from the cloned receptor, whereas the B2 receptor antagonist Hoe-140 (D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, where Thi is L-[3-(2-thienyl)alanyl], Tic is D-(1,2,3,4-tetrahydroisoquinolin-3-yl-carbonyl), and Oic is L-[(3aS, 7aS)-octahydroindol-2-yl-carbonyl]) does not. Therefore, the expressed receptor has the pharmacological characteristics of the B1 receptor subtype. The availability of both the cloned human B1 and B2 bradykinin receptors should allow the elucidation of the relative contributions of these two receptor subtypes in acute and chronic inflammatory processes.

Amino Acid Sequence↗

PubMed1994

Cloning, expression and characterization of human alpha adrenergic receptors alpha 1a, alpha 1b and alpha 1c.

The three human alpha adrenergic receptor subtypes, alpha 1a, alpha 1b, and alpha 1c, have been cloned, expressed in COS-7 cells, and characterized pharmacologically. Competition binding studies of several adrenergic ligands with the three human receptor subtypes reveals a distinct pharmacological profile for each receptor subtype. RNase protection analysis demonstrates that the three receptor subtypes have different patterns of distribution in human tissue. The availability of the three human alpha 1 receptor subtypes will facilitate the development of subtype-selective alpha 1 antagonists for a variety of therapeutic indications.

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

Characterization of the interaction of N-acyl-L-tryptophan benzyl ester neurokinin antagonists with the human neurokinin-1 receptor.

We have recently shown that a series of N-acyl-L-tryptophan benzyl esters are potent substance P antagonists (Macleod, A. M., Merchant, K. J., Cascieri, M. A., Sadowski, S., Ber, E., Swain, C. J., and Baker, R. (1993) J. Med Chem. 14, 2044-2045). We now report the detailed characterization of the interaction of N-acetyl-L-tryptophan-3,5-bistrifluoromethyl benzyl ester (L-732,138) with the human neurokinin-1 (NK-1) receptor. L-732,138 inhibits the binding of 125I-substance P to the cloned human NK1 receptor expressed in Chinese hamster ovary cells with an IC50 of 2.3 +/- 0.7 nM. In contrast, it has 200-fold lower affinity for the cloned rat NK-1 receptor and has > 1000-fold lower affinity for the human NK-2 and NK-3 receptors. L-732,138 acts as a competitive antagonist of substance P, as shown by functional Schild analysis of the inhibition of substance P-induced inositol phosphate synthesis, by kinetic analysis of the dissociation rate, and by thermodynamic analysis of the equilibrium binding of 125I-substance P to the NK-1 receptor. L-732,138 also competitively inhibits the binding of the quinuclidine amine antagonist, [125I]L-703,606, to the receptor. The compound has 230- and 10-fold reduced affinity for mutant NK-1 receptors in which histidine 265 or histidine 197, respectively, are replaced with alanine. We have previously shown that these residues play key roles in the binding of quinuclidine antagonists to the NK-1 receptor. These results suggest that the tryptophan and quinuclidine series of NK-1 antagonists bind to similar binding sites on the human NK-1 receptor.

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↗