Human Beta-2 Adrenergic Receptor
Created by Brendan Zotter
The Human Beta-2 Adrenergic Receptor (ADRB2) is an integral membrane protein found largely in smooth muscle tissue throughout the body. It is a member of the rhodopsin-like receptor family, which is characterized by the presence of seven membrane-spanning alpha helical domains(See Image 1). The ADRB2 is a G-protein coupled receptor (GPCR) that binds a family neurotransmitter ligands called Catecholamines, which include epinephrine and norepinephrine. These neurotransmitters are ubiquitous in the autonomic nervous system of vertebrate species, specifically within the sympathetic component, mediating fight-or-flight responses. Specifically, the ADRB2 is responsible for delaying digestion, delaying urination, and increasing respiration by relaxing the GI muscles, bladder wall muscles, and transverse bronchiolar muscles, respectively (Rang, 2007). Further, activation of the ADRB2 initiates vasodilation of vessels throughout the abdomen to increase blood flow, and therefore oxygen flow, to vital organs. Finally, ADRB2 activation also stimulates cardiac contractions, pupil dilation, and glycogenolysis in the liver (Rang, 2007).
Upon binding a ligand, the GB subunit of the ADRB2 G Protein dissociates and activates membrane-bound adenylyl cyclase (AC). AC converts AMP to cAMP, which activates Protein Kinase A (PKA). PKA phosphorylates multiple target proteins throughout the cell and induces a second-messenger cascade that amplifies the cellular response to the signal molecule (See Image 2). Since it activates an AC, it is designated as a Galpha-s subunit (Purves et al., 2008; Rasmussen et al., 2007).
The ADRB2 is approximately 46.459 kD in size and has an approximated isoelectric point of 6.59 (Gasteiger et al., 2003). It is very similar in primary structure to the Beta-2 Adrenoceptors found in several other organisms, with over 90% sequence similarity to receptors in Rabbit, Guinea Pig, Rhesus Macaque, and Chimpanzee(Uniprot, no PDB IDBs available), indicating a high level of conservation across species(Gasteiger et al., 2003). These homologous receptor proteins performed identicial chemical functions on the cellular level. Furthermore, the ADRB2 showed greater than 70% sequence similarity to Beta-1 Adrenoceptors isolated from the Turkey (PDB: 2VT4) (Jain et al., 2010). Activatation of the Beta-1 Adrenoceptor elicits similar system-wide sympathetic response, but instead most strongly activates salivary gland secretions and stimulates cardiac muscle contraction (Holm & Rosenstrom, 2010) In addition, greater than 30% primary structure similarity was reported with the human adenosine A2A receptor (PDB ID: 3EML)(Rang, 2007). The Human A2A is a GPCR that binds extracellular Adenosine and activates AC. Similar to the Adreneroceptors, the A2A is involved in the mediation of the sympathetic nervous system response. Specifically, stimulation of this receptor causes vasodilation of coronary arteries and the release of glutamate and dopamine in certain parts of the brain.
In terms of tertiary structure, the protein is in the rhodopsin superfamily, which includes many GPCRs, opsin proteins and chemokine receptor-like proteins. Specifically, the Adrenoceptors are in subfamily A17, which includes 5-hydroxytryptamine (5-HT) receptors, Dopamine Receptors, Histamine H2 Reptors, and Trace Amine Receptors(Holm & Rosenstrom, 2010). The B2ADR showed approximately 20% sequence similarity, but much tertiary structure similarity to several other Rhodopsin proteins, specifically Squid Rhodopsin (PDB ID: 2Z73)(Rang, 2007). Tertiary structure analysis on the DALI server showed 212 aligned residues and a Z-score of approximately 18 for this comparison (Holm & Rosenstrom, 2010). Both the human B2ADR and Squid Rhodopsin are large hydrophobic proteins that have seven membrane-spanning alpha-helices and are involved in transduction of extracellular signals across the cell membrane to activate a coupled G-Protein. The primary difference between the two is the nature of the activating ligand. The ligand is a catecholamine for B2ADRs, but a photon for Squid Rhodopsin. The photon interacts with a covalently bound chromophore cofactor called retinal, causing isomerization, and subsequently activating the associated G-Protein (Joost & Methner, 2002)
Its exact tertiary structure has been difficult to elucidate due to its instability in detergent solutions and its inherent structural variability, the latter attributed largely to its significant basal, agonist-independent activity level. The paradigm for modeling the tertiary structure of B2ARs, until only recently, was based on the known structure of bovine rhodopsin (PDB ID: 3C9L)due to its extreme abundance in bovine retina and its relative stability with the inverse agonist, 11-cis-retinal (Rasmussen et al., 2007). Critical structural and conformational differences between the two that make that model largely inaccurate have since been demonstrated and will be discussed.
In order to facilitate crystallographic study of the receptorBs structure, the B2AR was complexed with the inverse agonist carazolol and a stabilizing molecule was bound to the third intracellular loop. In the study performed by Rasmussen et al, the stabilizing molecule was a monoclonal rabbit-derived antibody, Mab5 (shown here with light chains in blue and heavy chains in orange) and in the study by Cherezov et al, a T4 Lysozyme was used. In both cases, care was taken to ensure that no alteration of the receptor conformation was induced by the presence of these stabilizing molecules(Rasmussen et al., 2007; Cherezov et al., 2007). B2AR is composed of seven membrane-spanning alpha-helices, with short connecting loops between them projecting into both the cytosolic and extracellular environments, along with two short non-spanning helices ( one near the C-Terminus found in all rhodopsin-like receptors, and one unique helix found in an extracellular loop). The seven helices of B2AR form a helical bundle, creating a deep, largely negatively-charged well (red area at center of image, image colored by residue charge) on the extracellular surface which contains the ligand-binding site. On the intracellular face of the bundle lies the positively-charged G-Protein interaction site (blue area at center of image, image colored by residue charge).It has been speculated that the receptor may exist as a dimer based on computer simulations of interactions between receptors, but no model of the dimerization interface or functional relevance of dimerization has been established (Cherezov et al., 2007).
With new crystallographic data, as well as mutagenesis studies, the amino acids interacting with both the ligand and the associated G-Protein have been determined with some certainty, as well as the topology of the ligand binding site. The lack of ligand-binding activity in the extracellular loops, as well as their somewhat simple structure, suggests that the ligand binding site sits not on the surface of the helical bundle, but deeper inside(Cherezov et al., 2007). As discussed earlier, rhodopsin binds a single retinal molecule permanently and only needs to allow a single photon to reach its ligand-binding domain. The B2AR must allow for relatively large catecholamine molecules to repeatedly travel to and from from the binding site. The relatively large extracellular loop between helices IV and V would interfere with this process. It is held clear of the binding site entrance by both an intra-loop disulfide bridge (between C184 and C190), as well as an inter-loop bridge to a neighboring domain (C-106 and C-191).(Cherezov et al., 2007). Additionally, the presence of an unexpected helical domain within this extracellular loop increases its rigidity and allows the disulfide bonds to anchor it away from the binding site without it buckling in the middle(Cherezov et al., 2007). The homologous structure in the same loop of bovine rhodopsin instead takes the form of several beta-sheets that sit over the retinal molecule, isolating it in a hydrophobic pocket. Herein lies a critical difference in the structures of these two molecules.
Within the ligand-binding site itself, mutagenesis studies and computer modeling suggest that critical interactions occur between the ligand and the following amino acids: D-113, V-114, W-286, F-289, F-290, and N-312(seen here in a ball and stick model and here in a space-filling model) (Rasmussen et al., 2007). Importantly, F-289 and F-290 form an extended conjugated aromatic system that causes a rotomeric change in a largely conserved W residue at position 286, with potential conformational implications for the receptor (as seen here in ball and stick and here in space-filling models). This pathway has been previously suggested for both rhodopsin and GPCR activation mechanisms, though not proven definitively (Schwartz et al., 2006). However, evidence has been found to suggest one possible mechanism governing the activity of the G-Protein activation domain of B2AR. There is a highly conserved (E/D)-R-(Y/W) sequence found in all rhodopsin molecules, as well as B2AR, which is referred to as the ionic lock. This consists of a system of H-Bonds between the DRY sequence and another E residue (R-131 and E-268 in B2AR (seen here in ball and stick and here in space-filling) and R-135 and E-247 in rhodopsin). When rhodopsin is activated by light, a conformational change moves these two amino acids farther apart (2.9 A to 4.1 A), weakening the H-Bond between them. In B2AR, these amino acids are already 6.2 A apart (similar to the activated form of rhodopsin), explaining its constitutive activity since the negative control exerted by this hydrogen bond structure is significantly weaker (Rasmussen et al., 2007).