A conserved helix 2 Asp is required for the proper function of many G-protein-coupled receptors. To reveal the structural basis for the role of this residue, the additive effects of mutations at this locus and at a conserved helix 7 locus were investigated in the 5-HT2A receptor. All mutant receptors studied retained high affinity agonist and antagonist binding. Whereas an Asp-->Asn mutation in helix 2 eliminated coupling, interchanging the residues at the two positions by a second mutation of Asn-->Asp in helix 7 restored receptor function. These data suggest that these residues are adjacent in space and interact. The loss of function observed with Ala at either position is consistent with each side chain forming hydrogen bonds. Molecular dynamics simulations were performed on three-dimensional computational models of agonist-receptor complexes of both the wild-type receptor and the Asp-->Asn mutant receptor. Consonant with the lack of coupling observed for the mutant construct, introducing the mutation into the computational model produced a conformational change in a direction opposite to that seen from computational simulations of activation of the wild-type receptor model. These results implicate both loci in a common hydrogen-bonding network underlying receptor activation by agonist.

An Arg present in the third transmembrane domain of all rhodopsin-like G-protein-coupled receptors is required for efficient signal transduction. Mutation of this Arg in the gonadotropin-releasing hormone receptor to Gln, His, or Lys abolished or severely impaired agoniststimulated inositol phosphate generation, consistent with Arg having a role in receptor activation. To investigate the contribution of the surrounding structural domain in the actions of the conserved Arg, an integrated microdomain modeling and mutagenesis approach has been utilized. The gonadotropin-releasing hormone (GnRH) 1 receptor is a member of the rhodopsin-like G-protein-coupled receptor (GPCR) family (1, 2). These heptahelical proteins include the visual opsins and various receptors for neurotransmitters, peptides, and glycoproteins. Activation of these receptors by their diverse agonists is associated with conformational changes in the receptor that facilitate a signal-propagating interaction with G-proteins (3). These conformational changes can involve relative movement of helices, as reported for rhodopsin (4, 5) and/or rotation of the helices as found in a constitutively active adrenergic receptor (6).Sequence alignment of GPCRs shows that certain amino acids are highly conserved at corresponding positions within the putative transmembrane domains (TMD) (7). Transitions among receptor conformations may reflect dynamic changes in side chain interactions within the receptor. Two of these conserved residues have been studied by reciprocal mutation in the GnRH and serotonin receptors, and the results suggest that the TMD 2 and 7 side chains have an interdependent role in receptor activation (8, 9). Most likely several other conserved side chains also interact to form the skeleton required for the conformational rearrangements that accompany the transition between inactive and active receptor states.The elucidation of the intramolecular interactions and conformational changes underlying receptor activation is hindered by the absence of high resolution structural data for any GPCR. The available low resolution projection maps of rhodopsin do not allow inferences about specific side chain interactions (10, 11). A prevalent approach to investigate structure-function relations of GPCRs is to introduce structural perturbations via site-directed mutagenesis and to evaluate their effect on receptor phenotype in binding and signal transduction assays (12). However, determining the phenotype of mutant receptors does not lead to an unequivocal interpretation concerning the structural basis of that phenotype (13).Molecular modeling has facilitated the integration of experimental observations and biophysical data into a mechanistic scheme for receptor structure and function (12,14). Structural and functional details of ligand binding (15,16) and receptor activation by agonist complexing (8,17) and by constitutively activating mutations (18) have been simulated in such models. The receptor models can thus provide a rationalization of current experime...

No abstract

The decapeptide gonadotropin-releasing hormone controls reproductive function via interaction with a heptahelical G protein-coupled receptor. Because of molecular model of the receptor predicts that Lys121 in the third transmembrane helix contributes to the binding pocket, the function of this side chain was studied by site-directed mutagenesis. Substitution of Arg at this position preserved high affinity agonist binding, whereas Gln at this position reduced binding below the limits of detection. Leu and Asp at this locus abolished both binding and detectable signal transduction. The EC50 of concentration-response curves for coupling to phosphatidyl inositol hydrolysis obtained with the Gln121 receptor was more than 3 orders of magnitude higher than that obtained for the wild-type receptor. In order to determine whether the increased EC50 obtained with this mutant reflects an altered receptor affinity, the effect of decreases in wild-type receptor density on concentration-response curves was determined by irreversible antagonism. Progressively decreasing the concentration of the wild-type receptor increased the EC50 values obtained to a maximal level of 2.4 +/- 0.2 nM. Comparison of this value with the EC50 of 282 +/- 52 nM observed with the Gln121 receptor mutant indicates that the agonist affinity for this mutant is reduced more than 100-fold. In contrast, antagonist had comparable high affinities for the wild-type, Arg121, and Gln121 mutants. The results indicate that a charge-strengthened hydrogen bond donor is required at this locus for high affinity agonist binding but not for high affinity antagonist binding.

Mutation of Asp(2.61(98)) at the extracellular boundary of transmembrane helix 2 of the gonadotropin-releasing hormone (GnRH) receptor decreased the affinity for GnRH. Using site-directed mutagenesis, ligand modification, and computational modeling, different side chain interactions of Asp(2.61(98)) that contribute to high-affinity binding were investigated. The conservative Asp(2. 61(98))Glu mutation markedly decreased the affinity for a series of GnRH analogues containing the native His(2) residue. This mutant showed smaller decreases in affinity for His(2)-substituted ligands. The loss of preference for His(2)-containing ligands in the mutant receptor shows that Asp(2.61(98)) determines the specificity for His(2). Analysis of the affinities of a series of position 2-substituted ligands suggests that a hydrogen bond forms between Asp(2.61(98)) and the delta NH group of His(2) and that Asp(2. 61(98)) forms a second hydrogen bond with the ligand. Substitution of Asp(2.61(98)) with an uncharged residue further decreased the affinity for all ligands and also decreased receptor expression. Computational modeling indicates an intramolecular ionic interaction of Asp(2.61(98)) with Lys(3.32(121)) in transmembrane helix 3. The uncharged, Lys(3.32(121))Gln mutation also markedly decreased agonist affinity. The modeling and the similar phenotypes of mutants with uncharged substitutions for Asp(2.61(98)) or Lys(3.32(121)) are consistent with the presence of this helix 2-helix 3 interaction. These studies support a dual role for Asp(2.61(98)): formation of an interhelical interaction with Lys(3.32(121)) that contributes to the structure of the agonist binding pocket and an interaction with His(2) of GnRH that helps stabilize agonist complexing.