Functional Microdomains in G-protein-coupled Receptors

Abstract

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...

RNA Editing of the Human Serotonin 5-HT2CReceptor Alters Receptor-mediated Activation of G13Protein

“…In this system, steps 1 and 3 are similar for each condition, so any shift in potency upon the addition of G ␣ subunits is a reflection of receptor/G-protein coupling. Previous R-SAT studies of the co-expression of muscarinic receptors with G q/11 (17), G 12 (16), G 13 (18), and G 14 and G 15 2 provided evidence that 1) these G-proteins are expressed under these experimental conditions and 2) efficient receptor/G-protein coupling can be detected by leftward shifts in agonist doseresponse curves.…”

“…Another consequence of RNA editing may be to alter the specificity of activation of heterotrimeric G-proteins and thereby shift intracellular signaling pathways. To test this hypothesis, we examined the ability of three 5-HT 2C R isoforms to functionally couple with the ␣ subunits of various heterotrimeric G-proteins of the G q family (G q , G 11 , G 14 , G 15 , and G 16 ) and the G 12 family (G 12 and G 13 ) using the cell-based functional assay, Receptor Selection/Amplification Technology (R-SAT).…”

A functional transmembrane complex: The luteinizing hormone receptor with bound ligand and G protein

“…Sitedirected mutagenesis was used to perform Ala-scanning of loop 2 and much of loop 3, followed by characterization of the wild type (WT) and mutant LHRs in transiently transfected HEK 293 cells. Extensive binding and signaling measurements permit determination of the coupling efficiency, Q, that relates receptor expression (B max ), hCG binding (K d or IC 50 ), and cAMP signaling (EC 50 and maximal response to a saturating dose of hCG, R max ) (Ballesteros et al, 1998).…”

Arrestin Binding to the G Protein-coupled N-Formyl Peptide Receptor Is Regulated by the Conserved “DRY” Sequence

“…Scheer et al (16) speculate that for the ␣ 1b -adrenergic receptor, mutations of the arginine residue can induce different states of the receptor. Ballesteros et al (29) suggest that a highly conserved aspartate, which is on helix 2 in most GPCRs and on helix 7 in the gonadotropin-releasing hormone receptor, forms a salt bridge with the arginine of the DRY sequence in the active state of the GPCR. This conflicts with the view by the Scheer et al (16), which argues that the arginine is not engaged in a salt bridge interaction.…”

“…The role of the DRY sequence in two other GPCRs, the gonadotropin-releasing hormone receptor (29) and the ␣ 1b -adrenergic receptor (16), has been extensively modelled. Work with both receptors suggests that the arginine in the DRY sequence plays a key structural role in receptor activation.…”