Probing the Binding Sites of GPCRs Using Unnatural Amino Acids: The Role of the Cation-π Interaction
3.1 Introduction
G-protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptor proteins in the human genome and constitute a prominent class of targets for the pharmaceutical industry(2-4). These receptors share a topology consisting of seven transmembrane helices. In 2007, of 324 molecular drug targets, 25%
of approved pharmaceuticals targeted GPCRs(5). This large proportion is due in part to the number and diversity of cellular pathways linked to GPCR activation. These receptors can be activated by a diverse array of stimuli such as neurotransmitters, peptides, odorants, proteins, lipids, and photons. The signaling pathways activated are involved in processes such as vision, olfaction, taste, memory, drug addiction, and the regulation of cardiac function (3, 6, 7). Accordingly, they have been studied extensively throughout academia and industry, utilizing the full range of chemical, biochemical, and biophysical techniques.
GPCR signaling begins with an extracellular stimulus such as ligand binding.
The binding of ligand induces a conformational change in the transmembrane helices and shifts the equilibrium from the inactive to the active receptor conformation. In the active conformation, the intracellular face of the receptor binds heterotrimeric G-proteins (α, β, and γ subunits)(8). The binding of G-protein facilitates the exchange of GDP for GTP in the Gα subunit and dissociation from the Gβγ subunits. The dissociated Gα and Gβγ subunits can then each act on numerous different cellular pathways. There are several different Gα subfamilies, and each one acts on different effectors. Gs activates adenyl
cyclase, Gi inhibits adenyl cyclase (AC), Gq modulates phospholipase C (PLCβ), G12/13
mediates Rho GTPase activity, and Gi/o gates G-protein activated inward rectifying potassium channels (GIRKs) and inhibits AC (9). Hydrolysis of GTP within the Gα subunit leads to reassociation of the Gα and Gβγ subunits and the termination of G protein signaling (figure 3.1).
Figure 3.1. GPCR signaling (figure courtesy of Dr. Michael Torrice).
The aminergic class of GPCRs respond to the monoamine neurotransmitters, such as epinephrine, acetylcholine, serotonin, and dopamine. These receptors belong to class A, which also includes rhodopsin and the olfactory receptors. In recent years the field has been energized by several high-resolution crystal structures of mammalian GPCRs
that build on the earlier, highly informative structural studies of rhodopsin and bacteriorhodopsin (10-15).
The ligand binding site of aminergic GPCRs is a crevice formed between the seven transmembrane helices (16, 17) (figure 3.2). Key elements of this site include a cluster of conserved aromatic residues and a highly conserved Asp on TM3, D3.32. This numbering uses the one-letter amino acid code, the helix number (1-7), and a residue index number. To index each residue of a helix, the most conserved residue in the helix is denoted as 50 and all other residues are numbered N-terminal to C-terminal accordingly. For example, D3.32 refers to an Asp residue on TM3, 18 residues in the N- terminal direction from the highly conserved Arg residue, R3.50. It is generally accepted that the negative charge of D3.32 interacts with the positively charged amine group of the aminergic ligands. Indeed, the 3 crystal structures of adrenergic receptors show the ligand interacting directly with D3.32(12-14).
Figure 3.2. Structure of the β2AR.
TM helices are labeled I through VIII. Bound inverse agonist Carazolol shown in red.
In receptors that bind catechol-containing agonists (dopamine and epinephrine) there is a set of conserved Ser residues on TM5 (S5.46 and S5.43). The hydroxyls of these serines are proposed to hydrogen bond with the two hydroxyls of the catechol moiety.
The structural snapshots provided by crystallography greatly enhance our understanding of specific receptors, but also raise many new issues. Key among these is the extent to which the information from available structures can be extrapolated to the hundreds of other GPCRs. In addition, a key goal in the study of GPCRs—and all receptors—is a description of the interconversions among several structural states that underlie the protein’s biological function. It can be a challenging task to deduce a signaling mechanism from static images. As such, structure-function studies, guided by the new structural advances, will remain an important tool in evaluating GPCR function and the nature of drug-receptor interactions in this family.
In recent years, unnatural amino acid mutagenesis on ion channels and receptors expressed in Xenopus oocytes has provided a powerful tool for uncovering crucial drug- receptor interactions and signaling mechanisms (18, 19). The method has been especially successful in highlighting the distinctions among related members of a receptor family and in providing insights into the extent to which structural studies of model systems directly apply to related receptors. GPCRs present an especially attractive target for unnatural amino acid mutagenesis, given the importance of the family, the significant
pharmacological variations among closely related family members, and the central role of structural rearrangements in their biological function.
Incorporating unnatural amino acids into GPCRs, however, presents unique challenges. Most unnatural amino acid mutagenesis studies in eukaryotic cells have focused on ion channels. These studies exploit the exquisite sensitivity of electrophysiology, which allows for detailed characterization even when only small quantities of protein are produced, as is often the case with unnatural amino acid mutagenesis. Because GPCRs are not ion channels and instead produce downstream signals though second messenger systems, a direct readout of GPCR activation during an unnatural amino acid experiment is not possible.
This chapter describes the first general strategy for chemical-scale studies of GPCRs using unnatural amino acid incorporation in a vertebrate cell. Electrophysiology again provides the functional readout, through downstream activation of a K+ channel.
Utilizing this system we investigated several key aromatic amino acids in the agonist- binding region of the D2 dopamine receptor and M2 muscarinic acetylcholine (ACh) receptor. We find that W6.48, a residue long postulated to play an important role in signaling, makes a cation-π interaction to dopamine in the active state of the D2 receptor.
Interestingly, ACh does not make the same interaction to the conserved W6.48 of the M2
receptor. We also identify F6.51 and F6.52 as critical residues in the activation of the D2 receptor by dopamine.
3.2 Results