Structure and function of G protein-coupled receptors using NMR spectroscopy

Nathans and Hogness [1] cloned the gene for the visual pigment rhodopsin in 1983, and found that the receptor contained seven hydrophobic membrane-spanning helices. At roughly the same time, the paradigm in the visual receptor field that rhodopsin functions as a light-activated Ca2+ channel was being overturned in favor of a competing model that rhodopsin is responsible for activating a heterotrimeric G-protein [2]. In 1986, a landmark paper by Strader, Lefkowitz and co-workers [3] reported the sequence of the β2-adrenergic receptor. They recognized that the β2-adrenergic receptor shares significant sequence homology with rhodopsin, including seven transmembrane (TM) segments. They proposed that the amine ligands of the adrenergic receptors intercalate into the TM helices and trigger receptor activation in the same manner as the retinal in rhodopsin. Because the receptors of the adenylate cyclase system were known to activate G-proteins [2,4,5], the similarity between the β2-adrenergic receptor and rhodopsin provided the first hint of the existence of a large G protein-coupled receptor (GPCR) family. Early structural work in the emerging GPCR field was guided by two pioneering studies. In 1993, Schertler, Villa and Henderson [6] reported an electron density projection map based on cryo-EM images of rhodopsin that showed the seven-helix architecture is distinctly different from that of bacteriorhodopsin, which had been a favored template for modeling GPCRs. In 1996, Hubbell and Khorana [7] used site-directed spin labeling in combination with EPR spectroscopy to reveal that activation is mediated by rigid body rotation of the TM helices. A breakthrough in the GPCR field came in 2000 with the determination of the rhodopsin crystal structure [8] (Fig. 1). The structure was solved of the dark, inactive conformation of the protein. The structure of the active state, however, has been more elusive. When illuminated, the crystals of rhodopsin crack and dissolve [9], presumably due to rigid body helix motions that accompany receptor activation. In 2007, the crystal structure of the β2-adrenergic receptor was solved with the inverse agonist carazolol bound in the active site [10]. The structure of this first ligand-activated GPCR confirmed that the overall seven-transmembrane helix architecture is similar to that of rhodopsin. Together the rhodopsin and β2-adrenergic receptor structures provide a high-resolution framework for understanding the structural features that are responsible for locking these receptors in the off conformation, and a starting point for investigating the mechanism of receptor activation. Fig. 1 Crystal structure of rhodopsin. The first crystal structure of a GPCR was obtained in 2000 by Palczewski and Okada [8]. Views of rhodopsin spanning the membrane bilayer (a) and from the extracellular (or intradiscal) surface of the membrane (b) show the ... In this review, we describe the progress that has been made using NMR spectroscopy to characterize the structure and dynamics of GPCRs in membrane environments. NMR methods have been used extensively to establish the structural changes occurring upon the activation of rhodopsin, as well as several ligand-activated receptors. We describe below the importance of these receptors in terms of their cell biology and pharmacology, and then outline the role that NMR can play in answering questions of structure and function. 1.1 Cell biology The 7-transmembrane helix GPCRs have evolved to recognize and transduce signals as diverse as light, Ca2+, small organic molecules and proteins. These receptors are found in both vertebrates and invertebrates, and are typically divided into six classes (Class A–F) based on sequence homology and functional similarity [11–13]. However, the classification is still open to debate. For example, on the basis of phylogeny, the human GPCRs have been divided into five families (Rhodopsin-like, Secretin, Adhesion, Glutamate, and Frizzled/Taste2) [14]. In this scheme, the Class A receptors correspond to the Rhodopsin-like family, but the Class B receptors are divided into the Secretin and Adhesion families. Nevertheless, in all classification schemes proposed to date, the lack of homology between classes or families suggests that nature has converged on the same seven transmembrane helix framework multiple times. The Class A (Rhodopsin-like family) receptors respond to the presence of diverse stimuli ranging from light absorption to the binding of various ligands, which include small molecule amines and hormones. Class B (Secretin and Adhesion families) receptors are activated by peptides of the glucagon hormone family [15,16]. The Class C (Glutamate family) GPCRs are comprised of the metabotropic glutamate receptors. These receptors are characterized by a large N-terminal ligand binding domain [17], which appears to be structurally homologous to the amino terminal domain of the ligand-gated ionotropic glutamate receptors in postsynaptic neuronal membranes [18]. Pheromones (e.g. α-factor) secreted by Saccharomyces cerevisiae bind to Class D GPCRs (e.g. STE2) during the mating process. Similar mechanisms are involved in the mating of several fungi [19]. Class E receptors have been implicated in the chemotactic migration of slime mold and can potentially be exploited as antifungal targets [20,21]. Class F (Frizzled/smoothened/taste2 family) contains receptors in the Wnt signaling pathway [14], which perform indispensable roles in embryonic development [22]. The Class A receptors are by far the most populated class of GPCRs. In the GPCR database there are over 20,000 Class A sequences (http://www.gpcr.org/). In humans, 952 of 1061 GPCRs identified in the human genome are in Class A. Of the 952 human Class A receptors, most (509) are olfactory receptors. The remaining Class A GPCRs are subdivided into 18 subfamilies including the well studied visual and small molecule amine receptors, as well as hormone and peptide receptors. Despite the breadth of this group, there exists a degree of sequence conservation among these receptors. Furthermore, the Class A receptors share similar intracellular proteins (e.g. protein kinases, arrestins) that mediate receptor desensitization.