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D Hill-Eubanks

Publications and source records attributed to D Hill-Eubanks.

5 recordsLinked to original sources
PubMed1996

Structure of a G-protein-coupling domain of a muscarinic receptor predicted by random saturation mutagenesis.

The third intracellular loop (i3) plays a critical role in the coupling of many receptors to G-proteins. In muscarinic receptor subtypes, the N- and C-terminal regions (Ni3 and Ci3) of this loop are sufficient to direct appropriate G-protein coupling. The relative functional contributions of all amino acids within Ni3 was evaluated by constructing libraries of m5 muscarinic receptors containing random mutations in Ni3 and screening them using high throughput assays based on ligand-dependent transformation of NIH 3T3 cells. In receptors that retained a wild type phenotype, the pattern of functionally tolerated substitutions is consistent with the presence of three turns of an alpha helix extending from the transmembrane domain. All of the amino acid positions that tolerate radical substitutions face away from a conserved hydrophobic face that ends with an arginine, and helix-disrupting proline substitutions were not observed. All of the mutant receptors with significantly compromised phenotypes had amino acid substitutions in residues predicted to form the hydrophobic face. Similar data from the Ci3 region (Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R. (1995) J. Biol. Chem. 270, 3141-3146) are consistent with the presence of a single helical turn extending from the transmembrane domain, with an alanine that defines G-protein affinity. Functionally critical residues of Ni3 and Ci3 are predicted to be in close proximity where they form the G-protein-coupling domain.

3T3 Cells↗
PubMed1995

Structure-function of muscarinic receptor coupling to G proteins. Random saturation mutagenesis identifies a critical determinant of receptor affinity for G proteins.

To derive structure-function relationships for receptor-G protein coupling, libraries were created of human m5 muscarinic acetylcholine receptors (m5) randomly mutated in the C-terminal region of the third intracellular loop. Functional receptors were identified based on their ability to amplify NIH 3T3 cells in a ligand-dependent manner. These receptors either had wild-type phenotypes (Group 1) or were functionally impaired (Group 2). No "activated receptors" were identified. Tolerated substitutions in Group 2 receptors were randomly distributed and frequently included prolines and glycines. In contrast, tolerated substitutions in Group 1 receptors exhibited a periodicity proximal to transmembrane domain 6 were proline and glycine substitutions were not observed. These observations are consistent with a short alpha-helical extension of the C-terminal region of the third intracellular loop from transmembrane domain 6. Mutations at Ala-441 were most commonly associated with impaired function of Group 2 receptors. Twelve point mutations at Ala-441 were tested, and all caused marked increases in EC50 values with little effect on maximal response or agonist binding affinity. These results indicate that Ala-441 is a key determinant of m5 receptor affinity for G proteins and exists within the structural context of a short alpha-helix.

3T3 Cells↗
PubMed1995

Pharmacology of a constitutively active muscarinic receptor generated by random mutagenesis.

We have isolated a mutant m5 muscarinic receptor that mediates robust functional responses in the absence of agonists. This constitutively active receptor was isolated from a library of receptors containing randomly introduced mutations in the sixth transmembrane domain and contains the substitutions serine 465 for tyrosine and threonine 486 for proline. Although these individual residues are not conserved in other G-protein-coupled receptors, they are predicted to be at the junction between the sixth transmembrane domain and the last extracellular loop. The mutant receptor (CAm5) was subjected to detailed pharmacological analysis. All of the antagonists tested (atropine, quinuclidinyl benzilate, N-methyl scopolamine, 4-diphenylacetoxy-N-methylpiperidine and pirenzepine) fully suppressed both the constitutive and agonist-induced activities of CAm5 revealing that these ligands are negative antagonists (inverse agonists). The potency of these ligands was similar at the mutant and wild-type receptors, suggesting that the antagonist binding site of this receptor is unchanged. The mutant had increased sensitivity to the agonists carbachol, arecoline, and McN-A-343 as measured both by functional response and by radioligand binding. These effects are explained and predicted by a model in which the primary effect of the mutations is to alter a spontaneous equilibrium existing between the active and inactive states of the receptor.

3T3 Cells↗
PubMed1993

Muscarinic acetylcholine receptor subtypes: localization and structure/function.

Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues. The distributions of these subtypes and their potential physiological roles are discussed. By use of molecular genetic manipulation of cloned muscarinic receptor cDNAs, the regions of muscarinic receptors that specify G-protein coupling and ligand binding have been defined in several recent studies. Overall, these studies have shown that amino acids within the third cytoplasmic loop of the receptors define their selectivities for different G-proteins and that multiple discontinuous epitopes contribute to their selectivities for different ligands. The residues that contribute to ligand binding and G-protein coupling are described, as well as the implied structures of these functional domains.

Animals↗