Evi Kostenis

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.

Functional Characterization of a Series of Mutant G Protein αq Subunits Displaying Promiscuous Receptor Coupling Properties

The N termini of two G protein α subunits, αq and α11, differ from those of other α subunits in that they display a unique, highly conserved six-amino acid extension (MTLESI(M)). We recently showed that an αq deletion mutant lacking these six amino acids (in contrast to wild type αq) 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 αq/11 are critical for constraining the receptor coupling selectivity of these subunits, this region of αq was subjected to systematic deletion and alanine scanning mutagenesis. All mutant αq constructs (or wild type αq 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 αq) 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 αqsubunits to couple to the Gs-coupled β2-adrenergic receptor. Additional experiments indicated that the functional promiscuity displayed by all investigated mutant αqconstructs was not due to overexpression (as compared with wild type αq), 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 αq/11 subunits forms a tightly folded protein subdomain that is critical for regulating the receptor coupling selectivity of these subunits.

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.

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.

Functional expression of M1, M3 and M5 muscarinic acetylcholine receptors in yeast

The goal of this study was to functionally express the three Gq‐coupled muscarinic receptor subtypes, M1, M3 and M5, 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 (Bmax <u200a5u2003fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M1, M3 and M5 muscarinic receptors resulted in dramatic increases in Bmax values (53–214u2003fmol/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 M1, M3 and M5 receptors were unable to productively interact with the endogenous yeast Gu2003protein α‐subunit, Gpa1p, or a Gpa1 mutant subunit that contained C‐terminal mammalian Gαs sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Gαq hybrid subunit containing C‐terminal mammalian Gαq sequence, indicating that the M1, M3 and M5 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 Gu2003proteins, should facilitate the functional expression of other classes of Gu2003protein‐coupled receptors in yeast.

Pharmacological profile of NOP receptors coupled with calcium signaling via the chimeric protein Gαqi5

In this study, the Gαqi5 protein was used to force the human nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor to signal through the Ca2+ pathway in CHO cells. [Ca2+]i levels were monitored using the fluorometer FlexStation II and the Ca2+ dye Fluo 4xa0AM. Concentration response curves were generated with a panel of full and partial agonists, while NOP antagonists were assessed in inhibition-response curves. The following rank order of potency of antagonists was measured:

Molecular Aspects of Vasopressin Receptor Function

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

naloxone, which is superimposable to literature findings. The rank order of potency of full and partial agonists is also similar to that obtained in previous studies with the exception of a panel of ligands (UFP-112, Ro 64-6198, ZP120, UFP-113) whose potency was relatively low in the Gαqi5–NOP receptor calcium assay. Interestingly, these NOP ligands are characterized by slow kinetic of interaction with the NOP receptor, as demonstrated by bioassay experiments. These results demonstrated that the FlexStation II–Gαqi5–NOP receptor calcium assay represents an adequate and useful screening for NOP receptor ligands, particularly for antagonists.