Biomedical subjects

E Kostenis

Publications and source records attributed to E Kostenis.

At least 19 recordsLinked to original sources

PubMed2001

Single amino acid substitutions and deletions that alter the G protein coupling properties of the V2 vasopressin receptor identified in yeast by receptor random mutagenesis.

To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.

Amino Acid Sequence↗

PubMed2001

Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice.

Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed2001

Muscarinic allosteric modulation: M2/M3 subtype selectivity of gallamine is independent of G-protein coupling specificity.

Among the five subtypes of muscarinic acetylcholine receptors, the sensitivity towards allosteric modulation is generally higher in M2 and M4 receptors that preferentially couple to inhibitory G-proteins of the Gi/o type than in M1, M3, and M5 that preferentially couple to stimulatory G-proteins such as Gq/11. We aimed to check whether the high allosteric sensitivity of the M2 receptor compared to M3 is related to the differential G-protein coupling preference. As the third intracellular loop (i3) is known to be the major determinant in receptor G-protein coupling specificity, we used wild-type M2 and M3 receptors and the related chimeric constructs with exchanged i3-loops, i.e., M2 containing M3-i3 (M2/M3-i3) and M3 containing M2-i3 (M3/M2-i3). The allosteric effect of the archetypal modulator gallamine on the dissociation and the equilibrium binding of [3H]N-methylscopolamine ([3H]NMS) was measured in membranes of mouse A9L cells stably expressing the wild-type and the chimeric receptors (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees C). The dissociation of [3H]NMS was monophasic under all conditions studied. Control values of t 1/2 were (means +/- SEM, n = 4-7): M2: 3.8 +/- 0.2 min, M2/M3-i3: 4.8 +/- 0.3 min, M3:43.3 +/- 4.2 min, M3/M2-i3: 41.1 +/- 3.6 min. At M2 receptors, 0.2 microM gallamine allosterically reduced the apparent rate constant of dissociation k-1 to 51 +/- 5% of the control value (n = 5). At M2/M3-i3 the allosteric potency of gallamine was not significantly changed (0.2 microM gallamine --> k-1 = 61 +/- 4%, n = 7). At M3, a 20-fold higher concentration was required for an equieffective allosteric action (10 microM gallamine --> k-1 = 51 +/- 5%, n = 5). The potency of gallamine at M3/M2-i3 was not increased compared with M3 receptors (10 microM gallamine --> k-1 = 73 +/- 2%, n = 4) but even significantly diminished. [3H]NMS equilibrium binding experiments revealed that neither the binding constants of gallamine at free receptor subtypes (pKA,M2: 7.57 +/- 0.04, n = 4; pKA,M3: 5.56 +/- 0.13, n = 3) nor the factors of negative cooperativity with [3H]NMS (alphaM2 = 31 +/- 1, alphaM3 = 3 +/- 0.4) were affected by the exchanged i3-loops (pKA,M2/M3-i3: 7.65 +/- 0.03, pKA,M3/M2-i2: 5.35 +/- 0.24, alphaM2/M3-i3= 30 +/- 2, alphaM3/M2-i2 = 3 +/- 0.7). In conclusion, the different sensitivities of M2 and M3 receptors towards allosteric modulation by gallamine are not related to the G-protein coupling specificity of the receptors.

Allosteric Regulation↗

PubMed2001

Is Galpha16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors?

Identification of natural ligands for orphan G-protein-coupled receptors will help expand the boundaries of physiology and pharmacology. Powerful approaches are needed that can pair biologically active ligands with their corresponding receptors. Many attempts have been made to set up universal screening schemes such that receptor activation by its cognate ligand is transduced into a common intracellular signal that is amenable to high-throughput screening analysis. One possibility that achieves such a 'universal assay' takes advantage of the promiscuous nature of the G-protein subunit Galpha16. However, a truly critical look at Galpha16 is still required. In this article, the strengths, weaknesses, problems and pitfalls that are associated with the use of Galpha16 will be discussed, and suggestions of how problems might be overcome with an optimized universal G-protein system will be proposed.

Animals↗

PubMed2001

Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast.

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.

Amino Acid Sequence↗

PubMed2000

Structure-activity relationships in a series of bisquaternary bisphthalimidine derivatives modulating the muscarinic M(2)-receptor allosterically.

Hexane-bisammonium-type compounds containing lateral phthalimide moieties are well-established ligands of the common allosteric binding site of muscarinic M(2) receptors. Previous structure-activity relationships (SAR) revealed two positively charged centers and two lateral phthalimide moieties in a defined arrangement to be essential of a high allosteric potency. The purpose of this study was to replace one carbonyl group of the phthalimides with hydrogens, hydroxy, alkoxy, phenyl, benzyl, and benzylidene groups in order to check the influence of these substituents on the allosteric activity in antagonist-linked receptors. The analysis of the quantitative SAR indicated that a high allosteric potency is related to a certain amount of rigidity as well as polarizibility and the ability to form hydrophobic interactions.

Allosteric Regulation↗

PubMed1999

Enhancement of D1 dopamine receptor-mediated locomotor stimulation in M(4) muscarinic acetylcholine receptor knockout mice.

Muscarinic acetylcholine receptors (M(1)-M(5)) regulate many key functions of the central and peripheral nervous system. Primarily because of the lack of receptor subtype-selective ligands, the precise physiological roles of the individual muscarinic receptor subtypes remain to be elucidated. Interestingly, the M(4) receptor subtype is expressed abundantly in the striatum and various other forebrain regions. To study its potential role in the regulation of locomotor activity and other central functions, we used gene-targeting technology to create mice that lack functional M(4) receptors. Pharmacologic analysis of M(4) receptor-deficient mice indicated that M(4) receptors are not required for muscarinic receptor-mediated analgesia, tremor, hypothermia, and salivation. Strikingly, M(4) receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses (as compared with their wild-type littermates) after activation of D1 dopamine receptors. These results indicate that M(4) receptors exert inhibitory control on D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are coexpressed at high levels. Our findings offer new perspectives for the treatment of Parkinson's disease and other movement disorders that are characterized by an imbalance between muscarinic cholinergic and dopaminergic neurotransmission.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1999

Bisquaternary ligands of the common allosteric site of M2 acetylcholine receptors: search for the minimum essential distances between the pharmacophoric elements.

Structurally diverse molecules, such as alcuronium, gallamine, and tubocurarine as well as W84 and WDUO, are known to interact allosterically with ligand binding to muscarinic M2 acetylcholine receptors. Preliminary molecular modeling studies revealed two positive charges in the middle and two lateral aromatic areas to be essential elements of a high allosteric potency. To find out the optimum distances between these pharmacophoric elements, a systematic variation of the spacer in the series of W84, WDUO, and IWDUO compounds was performed. The allosteric reduction of the rate of dissociation of the antagonist [3H]-N-methylscopolamine from porcine heart M2 receptors served as a test system. The minimal essential distance between the positive charges was found to be 10 A. The length of the peripheral spacers connecting the positive charge and the lateral aromatic moiety appears to depend on the chemical functionality; the peripheral spacers have to be long and flexible enough to position the aromatic skeletons in the spatial neighborhood of the alkane middle chain: in the case of an oxime ether containing peripheral spacer, six atoms are required, and in the case of an alkane chain, four carbon atoms are necessary to adopt the pharmacophoric S-shape conformation.

Allosteric Site↗

PubMed1999

Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice.

Members of the muscarinic acetylcholine receptor family (M1-M5) are known to be involved in a great number of important central and peripheral physiological and pathophysiological processes. Because of the overlapping expression patterns of the M1-M5 muscarinic receptor subtypes and the lack of ligands endowed with sufficient subtype selectivity, the precise physiological functions of the individual receptor subtypes remain to be elucidated. To explore the physiological roles of the M2 muscarinic receptor, we have generated mice lacking functional M2 receptors by using targeted mutagenesis in mouse embryonic stem cells. The resulting mutant mice were analyzed in several behavioral and pharmacologic tests. These studies showed that the M2 muscarinic receptor subtype, besides its well documented involvement in the regulation of heart rate, plays a key role in mediating muscarinic receptor-dependent movement and temperature control as well as antinociceptive responses, three of the most prominent central muscarinic effects. These results offer a rational basis for the development of novel muscarinic drugs.

Analgesia↗

PubMed1999

Structure-function analysis of muscarinic receptors and their associated G proteins.

Each member of the muscarinic receptor family (M1-M5) can interact only with a limited subset of the many structurally closely related heterotrimeric G proteins expressed within a cell. To understand how this selectivity is achieved at a molecular level, we have used the G(i/0)-coupled M2 and the Gq/11-coupled M3 muscarinic receptors as model systems. We developed a genetic strategy involving the coexpression of wild type or mutant muscarinic receptors with hybrid or mutant G protein alpha subunits to identify specific, functionally relevant receptor/G protein contact sites. This approach led to the identification of N- and C-terminal amino acids on alpha(q) and alpha(i) that are critical for maintaining proper receptor/G protein coupling. Moreover, several receptor sites were identified that are likely to be contacted by these functionally critical G alpha residues. To gain deeper insight into muscarinic receptor structure, we recently developed a cysteine disulfide cross-linking strategy, using the M3 muscarinic receptor as a model system. Among other structural modifications, this approach involves the removal of most native cysteine residues by site-directed mutagenesis, the insertion of three factor Xa cleavage sites into the third intracellular loop, and systematic 'reintroduction' of pairs of cysteine residues. Following treatment of receptor-containing membrane preparations with factor Xa and oxidizing agents, disulfide cross-linked products can be identified by immunoprecipitation and immunoblotting studies. This approach should greatly advance our knowledge of the molecular architecture of muscarinic and other G protein-coupled receptors.

Amino Acid Sequence↗

PubMed1998

Functional characterization of a series of mutant G protein alphaq subunits displaying promiscuous receptor coupling properties.

The N termini of two G protein alpha subunits, alphaq and alpha11, differ from those of other alpha subunits in that they display a unique, highly conserved six-amino acid extension (MTLESI(M)). We recently showed that an alphaq deletion mutant lacking these six amino acids (in contrast to wild type alphaq) was able to couple to several different Gs- and Gi/o-coupled receptors, apparently due to promiscuous receptor/G protein coupling (Kostenis, E., Degtyarev, M. Y., Conklin, B. R., and Wess, J. (1997) J. Biol. Chem. 272, 19107-19110). To study which specific amino acids within the N-terminal segment of alphaq/11 are critical for constraining the receptor coupling selectivity of these subunits, this region of alphaq was subjected to systematic deletion and alanine scanning mutagenesis. All mutant alphaq constructs (or wild type alphaq as a control) were coexpressed (in COS-7 cells) with the m2 muscarinic or the D2 dopamine receptors, two prototypical Gi/o-coupled receptors, and ligand-induced increases in inositol phosphate production were determined as a measure of G protein activation. Surprisingly, all 14 mutant G proteins studied (but not wild type alphaq) gained the ability to productively interact with the two Gi/o-linked receptors. Similar results were obtained when we examined the ability of selected mutant alphaq subunits to couple to the Gs-coupled beta2-adrenergic receptor. Additional experiments indicated that the functional promiscuity displayed by all investigated mutant alphaq constructs was not due to overexpression (as compared with wild type alphaq), lack of palmitoylation, or initiation of translation at a downstream ATG codon (codon seven). These data are consistent with the notion that the six-amino acid extension characteristic for alphaq/11 subunits forms a tightly folded protein subdomain that is critical for regulating the receptor coupling selectivity of these subunits.

Amino Acid Sequence↗

PubMed1998

Molecular aspects of vasopressin receptor function.

The molecular mechanisms governing the G protein coupling selectivity of different members of the vasopressin receptor family were studied by using a combined molecular genetic/biochemical approach. While the V1a and V1b vasopressin receptors are selectively linked to G proteins of the Gq/11 class, the V2 vasopressin receptor is preferentially coupled to Gs. Systematic functional analysis of V1a/V2 hybrid receptors showed that the second intracellular loop of the V1a receptor is required and sufficient for efficient coupling to Gq/11, whereas the third intracellular loop of the V2 receptor is required and sufficient for coupling to Gs. By using a strategy involving the coexpression of the wild type V1a receptor with chimeric G protein alpha s/alpha q subunits, two C-terminal alpha q/11 residues were identified that are critical for proper receptor recognition. We previously demonstrated -in transiently transfected COS-7 cells- that selected mutant V2 vasopressin receptors (all of which have been identified in X-linked nephrogenic diabetes insipidus patients) containing inactivating mutations in the C-terminal third of the receptor protein (including missense, frameshift, or nonsense mutations) can be functionally rescued by coexpression with a C-terminal V2 receptor fragment (V2-tail) spanning the region where the various mutations occur. Co-immunoprecipitation experiments and a newly developed sandwich ELISA revealed that the V2-tail polypeptide directly interacts with the mutant V2 receptors thus creating a functional receptor protein. To study the potential therapeutic usefulness of these findings, CHO cell lines stably expressing low levels of functionally inactive mutant V2 vasopressin receptors (E242stop, Y280C, and W284stop) were created and infected with a recombinant adenovirus coding for the V2-tail polypeptide. Following adenovirus infection, arginine vasopressin (AVP) gained the ability to stimulate cAMP formation in all CHO cell clones studied. Adenovirus-mediated gene transfer also proved to be a highly efficient method to achieve expression of the V2-tail fragment (as well as of the wild type V2 vasopressin receptor) in MDCK renal tubular cells. We therefore speculate that the targeted expression of receptor fragments in vivo may represent a novel strategy in the treatment of human diseases caused by inactivating mutations in distinct G protein-coupled receptors.

Amino Acid Sequence↗

PubMed1997

Genetic analysis of receptor-Galphaq coupling selectivity.

Many different G protein-linked receptors are preferentially coupled to G proteins of the Gq/11 family. To elucidate the molecular basis underlying this selectivity, different Gq/11-coupled receptors (m3 muscarinic, V1a vasopressin, and gastrin-releasing peptide receptor) were coexpressed (in COS-7 cells) with mutant alphas subunits in which residues present at the C terminus of alphas were replaced with the corresponding alphaq/11 residues. Remarkably, whereas none of the receptors was able to interact with wild type alphas to a significant extent, all three receptors gained the ability to productively couple to a mutant alphas subunit containing a single Glu --> Asn point mutation at position -3. Moreover, the m3 muscarinic and the V1a vasopressin receptors but not the GRP receptor also gained the ability to interact with a mutant alphas subunit containing a single Gln --> Glu point mutation at position -5, indicating that the alphaq/11 residues present in these mutant G protein constructs play key roles in determining the selectivity of receptor recognition. To identify the site(s) on Gq/11-coupled receptors that can functionally interact with the C terminus of alphaq/11 subunits, we next analyzed the ability of a series of hybrid m2/m3 muscarinic receptors to interact with a mutant alphas subunit (sq5) in which the last five amino acids of alphas were replaced with the corresponding alphaq/11 sequence. Similar to the wild type m2 and m3 muscarinic receptors, none of the investigated hybrid receptors was able to efficiently interact with wild type alphas. Interestingly, however, three mutant m2 receptors in which different segments of the second and third intracellular loops were replaced with the corresponding m3 receptor sequences were identified, which, in contrast to the Gi/o-coupled wild type m2 receptor, gained the ability to efficiently activate the sq5 subunit. This observation suggests that multiple intracellular receptor domains form a binding pocket for the C terminus of G protein alphaq/11 subunits.

Amino Acid Sequence↗

PubMed1997

The N-terminal extension of Galphaq is critical for constraining the selectivity of receptor coupling.

Characteristically, an individual member of the superfamily of G protein-coupled receptors can interact only with a limited number of the many structurally closely related G protein heterotrimers that are expressed within a cell. Interestingly, the N termini of two G protein alpha subunits, Galphaq and Galpha11, differ from those of other alpha subunits in that they display a unique, highly conserved six-amino acid extension. To test the hypothesis that this sequence element is critical for proper receptor recognition, we prepared a Galphaq deletion mutant (-6q) lacking these first six amino acids. The -6q construct (or wild type Galphaq as a control) was coexpressed (in COS-7 cells) with several different Gi/o- or Gs-coupled receptors, and ligand-induced increases in inositol phosphate production were determined as a measure of G protein activation. Whereas these receptors did not efficiently interact with wild type Galphaq, most of them gained the ability to productively couple to -6q. Additional experiments indicated that the observed functional promiscuity of -6q is not due to overexpression (as compared with wild type Galphaq) or to a lack of palmitoylation. We conclude that the N-terminal extension characteristic for Galphaq/11 proteins is critical for constraining the receptor coupling selectivity of these subunits, indicative of a novel mechanism by which the fidelity of receptor-G protein interactions can be regulated.

Amino Acid Sequence↗

PubMed1997

Molecular basis of receptor/G protein coupling selectivity studied by coexpression of wild type and mutant m2 muscarinic receptors with mutant G alpha(q) subunits.

The molecular basis of receptor/G protein coupling selectivity was studied by using the m2 muscarinic receptor, a prototypical G(i/o)-coupled receptor as a model system. We could recently show that the m2 receptor can efficiently interact with mutant G protein alpha(q) subunits in which the last five amino acids were replaced with alpha(i2) or alpha(o) sequence [Liu, J., Conklin, B. R., Blin, N., Yun, J., & Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642-11646]. Additional mutagenesis studies led to the identification of a four-amino-acid motif on the m2 receptor (Val385, Thr386, Ile389, and Leu390) that is predicted to functionally interact with the C-terminal portion of alpha(i/o) subunits. To further investigate the structural requirements for this interaction to occur, these four m2 receptor residues were replaced, either individually or in combination, with the corresponding residues present in the G(q/11)-coupled muscarinic receptors (m1, m3, and m5). The ability of the resulting mutant m2 receptors to interact with a mutant alpha(q) subunit (qo5) in which the last five amino acids were replaced with alpha(o) sequence was investigated in co-transfected COS-7 cells [studied biochemical response: stimulation of phosphatidyl inositol (PI) hydrolysis]. Our data suggest that the presence of three of the four targeted m2 receptor residues (Val385, Thr386, and Ile389) is essential for efficient recognition of C-terminal alpha(i/o) sequences. To study which specific amino acids within the C-terminal segment of alpha(i/o) subunits are critical for this interaction to occur, the wild type m2 receptor was co-expressed with a series of mutant alpha(q) subunits containing single or multiple alpha(q) --> alpha(i1,2) point mutations at their C-terminus. Remarkably, the wild type m2 receptor, while unable to efficiently stimulate wild type alpha(q), gained the ability to productively interact with three alpha(q) single-point mutants, providing the first example that the receptor coupling selectivity of G protein alpha subunits can be switched by single amino acid substitutions. Given the high degree of structural homology among different G protein-coupled receptors and among different classes of G protein alpha subunits, our results should be of broad general relevance.

Amino Acid Sequence↗

PubMed1997

Structural basis of receptor/G protein coupling selectivity studied with muscarinic receptors as model systems.

Different muscarinic acetylcholine receptor subtypes were used as model systems to study the structural basis of receptor/G protein coupling selectivity. Extensive mutagenesis studies have previously led to the identification of single amino acids on the m3 muscarinic receptor protein (located in the second intracellular loop (i2) and at the N- and C-terminus of the third intracellular loop (i3)) that dictate selective recognition of Gq/11 proteins by this receptor subtype. Based on these results, we proposed a model of the intracellular m3 receptor surface in which the functionally critical residues project into the interior of the transmembrane receptor core. To identify specific regions on the G protein(s) that are contacted by these different, functionally critical receptor sites, we recently employed a novel experimental strategy involving the coexpression of hybrid m2/m3 muscarinic receptors with hybrid G alpha-subunits. Using this approach, we could demonstrate that the C-terminus of G protein alpha i/o-subunits is recognized by a short sequence element in the m2 muscarinic receptor ("VTIL") that is located at the junction between the sixth transmembrane domain (TM VI) and the i3 loop. We could show that this interaction is critically involved in determining coupling selectivity and triggering G protein activation. By using a similar strategy (coexpression of mutant muscarinic receptors with hybrid G alpha-subunits), other major receptor/G protein contact sites are currently being identified. These studies, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.

Animals↗

PubMed1996

Evidence for a multiple binding mode of bispyridinium-type allosteric modulators of muscarinic receptors.

The ligand binding properties of muscarinic receptors can be modulated by allosterically acting compounds. Here, a set of novel bispyridinium-type compounds was investigated which were designed to study structure-activity relationships and to provide more insight into the molecular events underlying the allosteric delay of the dissociation of [3H]N-methylscopolamine from muscarinic M2 receptors in porcine cardiac membranes. The parent compound, a non-substituted bispyridinium oxime, displayed a weak allosteric potency and was unable to prevent radioligand dissociation at maximum concentrations. Introduction of either a phthalimidomethyl-moiety or a dichlorobenzyl-moiety at one end of the parent compound led to a considerable increase of the allosteric activity with regard to both the potency and the maximum effect. In these unilaterally ring-substituted bispyridiniums, homologous contralateral non-aromatic modifications were accompanied by divergent potency shifts depending on whether the unilateral ring was phthalimidomethyl or dichlorobenzyl. The findings point to a multiple binding mode of bispyridinium compounds at M2 receptors in the [3H]N-methylscopolamine-occupied state, i.e., different orientations of the compounds at the allosteric binding area or even an interaction with distinct allosteric recognition sites.

Allosteric Regulation↗

PubMed1996

Two-point kinetic experiments to quantify allosteric effects on radioligand dissociation.

Allosteric modulation of a receptor may be proved experimentally by demonstrating an altered radioligand dissociation in the presence of the allosteric modulator. Two-point kinetic experiments provide a screening-type approach to determine the delay of radioligand dissociation caused by allosteric modulation. In this article, Evi Kostenis and Klaus Mohr describe a pitfall in the data analysis that may lead to a suboptimum determination of the allosteric potency in the case of monophasic dissociations, and suggest how this problem may be resolved.

Allosteric Regulation↗