Characterization of the Residues in Helix 8 of the Human β1-Adrenergic Receptor That Are Involved in Coupling the Receptor to G Proteins

Lidia A Gardner,Suleiman W Bahouth,Noel M Delos Santos,Stephen W White

doi:10.1074/jbc.m508500200

Abstract

Several key amino acids within amphipathic helix 8 of the human beta1-adrenergic receptor (beta1-AR) were mutagenized to characterize their role in signaling by G protein-coupled receptors. Mutagenesis of phenylalanine at position 383 in the hydrophobic interface to histidine (F383H) prevented the biosynthesis of the receptor, indicating that the orientation of helix 8 is important for receptor biosynthesis. Mutagenesis of aspartic acid at position 382 in the hydrophilic interface to leucine (D382L) reduced the binding and uncoupled the receptor from G protein activation. Mutagenesis of the basic arginine residue at position 384 to glutamine (R384Q) or to glutamic acid (R384E) increased basal and agonist-stimulated adenylyl cyclase activities. R384Q and R384E displayed features associated with constitutively active receptors because inverse agonists markedly reduced their elevated basal adenylyl cyclase activities. Isoproterenol increased the phosphorylation and promoted the desensitization of the Gly389 or Arg389 allelic variants of the wild type beta1-AR but failed to produce these effects in R384Q and R384E, because these receptors were maximally phosphorylated and desensitized under basal conditions. In contrast to the membranous distribution of the wild type beta1-AR, R384Q and R384E were localized mostly within intracellular punctate structures. Inverse agonists restored the membranous distribution of R384Q and R384E, indicating that they recycled normally when their constitutive internalization was blocked by inverse agonists. These data combined with computer modeling of the putative three-dimensional organization of helix 8 indicated that the amphipathic character of helix 8 and side chain projections of Asp382 and Arg384 within the hydrophilic interface might serve as a tethering site for the G protein.

Highlights

G protein-coupled receptors (GPCR) for mediating the functions of catecholamines
The ␤1-AR is a prominent member of class A GPCR, which are structurally related to the visual receptor rhodopsin that propagates its signal through the activation of the G protein transducin [1]
Based upon the rhodopsin model, it is expected that the corresponding hydrophobic amino acids in the human ␤1-AR, Phe383 and Phe387, will be buried in the hydrophobic core of the helix facing the inner leaf of the membrane, whereas the charged polar groups Arg384, Lys385, and Gln388 would cluster on the other side of the ␣-helix that is exposed to the cytoplasmic environment

Summary

EXPERIMENTAL PROCEDURES

Construction of FLAG-tagged Wild Type and Mutant ␤1-AR—cDNA encoding the Gly389 variant of the human WT ␤1-AR [15] was a generous gift from R. Each saturation and competition experiment was in triplicate, and each was replicated between three and five times to determine the means Ϯ S.E. Cyclic AMP Accumulation and Adenylyl Cyclase Assays—Transiently transfected cells in 6-well plates were switched to DMEM ϩ 25 mM HEPES for 2 h. Acid Strip Confocal Recycling Microscopy Protocol—HEK-293 cells expressing the FLAG-tagged WT ␤1-AR and its helix 8 point mutants or the pCMV-Tag vector (Stratagene) were grown on poly L-lysinecoated glass coverslips and serum-starved at 37 °C for 1 h in DMEM supplemented with 25 mM HEPES, pH 7.4. Ligand binding properties of isoproterenol at the wild type and helix 8 ␤1-AR mutants [125I]ICYP binding was determined as described under “Experimental Procedures” on membranes derived from HEK-293 cells expressing the wild-type ␤1-AR (Gly389 or Arg389) or point mutants of helix 8. A value of p Ͻ 0.05 was considered statistically significant

RESULTS

12 Ϯ 2a 31 Ϯ 4b 32 Ϯ 4c

DISCUSSION