β2 adrenergic receptor
β adrenergic receptor (AR) is a G protein coupled receptor (GPCR), also known as a seven transmembrane helix protein, in which epinephrine and norepinephrine released after sympathetic nerve stimulation bind as ligands. β adrenergic receptor has three subtypes, β1-, β2- and β3 ARs. Their tissue distribution and physiological function are distinct; β1 AR, mainly localized in cardiac myocyte, increase heart rate and enforce contraction, β2 AR, mainly localized in smooth muscle, relaxes smooth muscle, and β3 AR, mainly localized in adipose tissues and bladder, enhances lipolysis and urination. Although these function have been considered to mediate receptor–Gs coupling, recent studies suggest that β ARs can couple with multiple heterotrimeric G proteins; β1 AR couples with Gs and Gi/o, β2 AR with Gs, Gi/o with Gq, and β3 AR with Gs and Gi/o.
Despite extensive efforts, structural information of GPCR has been restricted to bovine photoreceptor protein, rhodpsin (xPSSS:2I35) for a long time. A research group of Kobilka and Stevens revealed the structure of human β2 AR on 2007. This is the first structure of ligand binding GPCR.
Fig.1 Overall structure
β2 AR is a membrane protein composed of seven transmembrane helices forming a helical bundle. In addition to the transmembrane helices, β2 AR has three extracellular loops, three intracellular loops, and an helix VIII facing inner leaflet of plasmamenbrane. A partial inverse agonist, which decreases the basal activity, carazolol binds and flexible intracellular third loop is replaced by a T4 lysozyme to stabilize the protein conformation.
Fig.2 Cholesterol mediated dimeric structure
Many GPCR including β2 AR are reported to function as homo- or hetero dimer. Homodimeric β2 AR with four cholesterol molecules on the interface of each subunit was formed when the crystal was grown with cholesterol. The cholesterol mediated dimer may be related the cholesterol dependent β2 AR function; for example in neonatal cardiac myocyte, activation of β2 AR results in sequential coupling to Gs and Gi while β2 AR couples only to Gs after depletion of cholesterol.
Fig.3 Comparison of the extracellular region of rhodpsin (left) and β2 AR (right)
The extracellular region of GPCR plays an important role on regulating ligand binding site located inside the helix bundle. Comparison of the extracellular region of rhodpsin and β2 AR revealed their difference. The second extracellular loop (ECL2) of rhodpsin forms β sheet buried in the helix bundle. On the other hand, The ECL2 of β2 AR forms short α helix exposed to the extracellular region. The exposed ECL2 of β2 AR is stabilized by two disulfide bonds (Cys184-Cys190 and Cys191-Cys106).
Fig.4 Comparison of ligand binding site of rhodpsin (left) and β2 AR (right)
The ligand binding sites of both rhodpsin and β2 AR locates aromatic residue-rich region within the helix bundle. In rhodpsin, the full inverse agonist, which suppresses all activity, of rhodpsin, 11-cis retinal, is sandwiched between Phe212 and Tyr268 and directly interacts with Trp265 via β ionone ring. Trp265 of rhodpsin is the key residue in stabilization of inactive state because the residue changes the orientation after photoactivation. In β2 AR, on the other hand, carazolol locates near the phe208, Phe289, Phe290 and Trp286. Although Trp286 of β2 AR corresponds to Trp265 of rhodpsin, carazolol does not directly interact with Trp286. Otherwise, carazolol binding to Phe290 stabilizes the interaction between Phe290 and Trp286, mimicking 11-cis retinal–Trp286 interaction in rhospsin.
Protein Data Bank (PDB)
author: Daisuke Ino