Mutational and Computational Analysis of the α1b-Adrenergic Receptor

Abstract

To investigate their role in receptor coupling to G q , we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the ␣ 1b -adrenergic receptor (AR) GPCRs are structurally characterized by seven transmembrane ␣-helices connected by alternating extracellular (e) and intracellular (i) loops. While the extracellular portion of these receptors is primarily involved in ligand binding, the cytosolic loops mediate the interaction of the receptors with a number of signaling and regulatory proteins, including G proteins, arrestins, and G protein-coupled receptor kinases (reviewed in Ref..2).Evidence suggests that a conformational adjustment within the helical bundle of the receptor underlies the process of agonist-induced activation of GPCRs (reviewed in Ref.3). The current hypothesis is that the transition from the inactive (R) to active (R*) state of a GPCR results in receptor interaction with, and activation of, a G protein. Thus a GPCR-mediated biological response involves a series of events (i.e. receptor activation, receptor-G protein interaction, and receptor-induced G protein activation) for which a detailed mechanism still remains elusive at the molecular level. Although residues located in the helical bundle and at the boundary between the membrane and the cytosol may play a role in the "conformational switch" underlying receptor activation, amino acids in the intracellular loops are believed to be more directly involved in receptor-G protein interaction and/or receptor-induced G protein activation. The combination of these two latter events, which cannot be unequivocally separated experimentally, is generally indicated with the term of receptor-G protein coupling.We have previously provided evidence that the negatively and positively charged amino acids of the conserved DRY motif at the cytosolic end of helix 3 play a key role in the activation process of the ␣ 1b -AR (4 -6). Following a combination of experimental and computer-simulated mutagenesis of the ␣ 1b -AR, we have hypothesized that protonation of the aspartate (Asp 142 ) and a shift of the arginine (Arg 143 ) out of a conserved "polar pocket" are crucial steps in the transition of the receptor from the inactive (R) to active (R*) state (4 -6).Several studies have tried to identify the amino acids of different GPCRs involved in G protein coupling at both experimental (as reviewed in Ref.2) and theoretical levels (7-10). The majority of these studies indicate that sequences in the i2 loop as well as in the N and C termini of the i3 loop play an

Identification of Cytoplasmic Domains of hVPAC1 Receptor Required for Activation of Adenylyl Cyclase

“…This supports the idea that IL2 in the hVPAC1 receptor is not important for receptor folding and function including coupling to G s . This contrasts markedly with the important role of the IL2 in class I receptors, which appear to function as a hVPAC1 Receptor Cytoplasmic Domainsswitch enabling G protein activation (27,28). In particular the (D/E)RY sequence located close to the junction of TM3 with IL2 is intimately involved in G protein activation of class I GPCRs (29).…”

Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins

“…1, B and C, Table I). Previous studies show that mutation of hydrophobic amino acids in intracellular loop 2 of the ␣ 1b -adrenoreceptor can eliminate agonist-mediated signal transduction (27). We, thus, generated a fusion protein between L151D ␣ 1b -adrenoreceptor and G 11 ␣.…”

“…These fragments were purified and ligated into pcDNA 3 vector (Invitrogen) previously digested with HindIII/XhoI. The choice of intracellular loop 2 Leu to Asp mutants was based on the studies of Greasley et al (27). For co-immunoprecipitation and tr-FRET studies, c-Myc (EQKLISEEDL) or FLAG (DYKDDDDK) epitopes were introduced immediately after the N-terminal methionine.…”

A Model for Constitutive Lutropin Receptor Activation Based on Molecular Simulation and Engineered Mutations in Transmembrane Helices 6 and 7

“…The theoretical model suggests that the stabilizing effect of Asp-542(6.30) is more likely exerted via an interhelical salt bridge rather than via an intrahelix H-bonding interaction as very recently suggested (41). It is noteworthy that, in addition to rhodopsin, a salt bridge between the amino acids homologous to Arg-442(3.50) and Asp-542(6.30) in LHR has recently been suggested as one of the intramolecular constraints stabilizing the inactive states of the ␣ 1b -adrenergic, ␤ 2 -adrenergic, and 5HT2A-serotoninergic receptors (37,(42)(43)(44)(45).…”