G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signaling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor (β2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signaling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino and carboxyl terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an alpha helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the alpha helical domain of Gαs relative to the ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signaling by a GPCR.

G protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β2-adrenergic receptor (β2AR), a prototypical GPCR. We labeled β2AR with 13CH3ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β2AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β2AR’s ability to engage multiple signaling and regulatory proteins.

G protein coupled receptors (GPCRs) are seven transmembrane proteins that mediate the majority of cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs1,2,3,4,5 reveal structural conservation extending from the orthosteric ligand binding site in the transmembrane core to the cytoplasmic G protein coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse, and therefore represents an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the β2 adrenergic receptor: a salt bridge linking extracellular loops (ECLs) 2 and 3. Small molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G protein activation (agonist, neutral antagonist, and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide new insight into the dynamic behavior of GPCRs not addressable by static, inactive-state crystal structures.

Emerging pathological evidence indicates that major chronic aging-related diseases such as atherosclerosis, arthritis, dementia, osteoporosis, and cardiovascular diseases, are inflammation-related. In this review, inflammation is examined as a possible underlying basis for the molecular alterations that link aging and age-related pathological processes. A proposal for the molecular inflammation hypothesis of the aging views the redox derangement that occurs during aging as the major factor for increased risk for age-related inflammation. Accumulated data strongly indicate the activation of redox-sensitive transcription factors and dysregulated gene expression under the age-related oxidative stress seems to be the major culprits. Key players involved in the inflammatory process are the age-related upregulation of NF-kappaB, IL-1beta, IL-6, TNFalpha, cyclooxygenase-2, adhesion molecules, and inducible NO synthase. Furthermore, data are presented on the molecular events involved in age-related NF-kappaB activation and phosphorylation by IkappaB kinase/NIK and MAPKs. Experimental data on anti-aging calorie restriction (CR) for its antiinflammatory efficacy by suppressing the upregulated proinflammatory mediators will be reviewed. Also, the involvement of another super family of transcription factors, PPARs (PPARalpha, gamma) as regulators of proinflammatory responses and NF-kappaB signaling pathway is described as well as a discussion on the physiological significance of a well-maintained balance between NF-kappaB and PPARs.

The stability of proteins that constitute the neurofibrillary tangles and senile plaques of Alzheimer disease suggests that they would be ideal substrates for nonenzymatic glycation, a process that occurs over long times, even at normal levels of glucose, ultimately resulting in the formation of advanced glycation end products (AGEs). AGE-modified proteins aggregate, and they generate reactive oxygen intermediates. Using monospecific antibody to AGEs, we have colocalized these AGEs with paired helical filament tau in neurofibrillary tangles in sporadic Alzheimer disease. Such neurons also exhibited evidence of oxidant stress: induction of malondialdehyde epitopes and heme oxygenase 1 antigen. AGErecombinant tau generated reactive oxygen intermediates and, when introduced into the cytoplasm of SH-SY5Y neuroblastoma cells, induced oxidant stress. We propose that in Alzheimer disease, AGEs in paired helical filament tau can induce oxidant stress, thereby promoting neuronal dysfunction.Proteins or lipids exposed to reducing sugars undergo nonenzymatic glycation and oxidation, initially with formation of Schiff bases and Amadori products on free amino groups, which ultimately undergo molecular rearrangement, to form irreversible advanced glycation end products (AGEs; refs. [1][2][3][4][5]. The AGEs are heterogeneous compounds of yellowbrown color and characteristic fluorescence (1-5). Accumulation of AGEs occurs on both intra-and extracellular structures, especially those whose turnover is prolonged. Although the formation of AGEs is accelerated in diabetes, it also occurs in normal aging. Proteins with many free amino groups (i.e., with high lysine content) are most readily glycated. AGE-modified proteins form crosslinks which result in aggregation and insolubility; they are also a continuing source ofpotentially damaging reactive oxygen intermediates (ROIs) and, when present extracellularly, interact with a distinct class of receptors (1-9). In cells, we have found that AGEs impart an oxidant stress manifested in endothelium by induction of heme oxygenase, activation of the transcription factor NF-KB, and formation of malondialdehyde epitopes of lipid peroxidation products (9). These perturbations, which result in changes in a spectrum of cellular properties (e.g., cell adherence, proliferation), were not accompanied by diminished cell viability (in short-term experiments), in keeping with a role for low levels of ROIs in signal transduction.The longstanding protein aggregates in Alzheimer disease (AD), such as paired helical filament (PHF) tau and amyloid .3protein (10)(11)(12) METHODS AGE ELISA, Immunoblotting, and Immunohistohemistry. AGE antigen was determined by using affinity-purified antibody to AGEs (9,13). This antibody selectively recognizes AGE forms of multiple proteins, but not the nonglycated counterparts (9) or formylated, maleylated, oxidized, or acetylated protein (9). To assay for AGE antigen (9), an ELISA was established by coating plates with brain homogenates/PHF tau (10-100 gg/ml) ove...

Salmeterol is a partial agonist for the β 2 adrenergic receptor (β 2 AR), and the first long-acting β 2 AR agonist (LABA) to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol has been controversial both for its safety and mechanism of action. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound β 2 AR in complex with an active-state stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between β 1 AR and β 2 AR explain the high receptor subtype selectivity. A structural comparison with the β 2 AR bound to the full agonist epinephrine reveals differences in the hydrogen bond network involving residues Ser 204 5.43 and Asn 293 6.55 . Mutagenesis and biophysical studies suggest that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G protein activation and the limited β-arrestin recruitment for salmeterol.

The human pathogen Pseudomonas aeruginosa coordinates the expression of virulence factors using quorum sensing, a signaling cascade triggered by the activation of signal receptors by small molecule autoinducers. These homoserine lactone autoinducers stabilize their cognate receptors and activate their functions as transcription factors. As quorum sensing regulates the progression of infection and host immune resistance, significant efforts have been devoted towards the identification of small molecules that disrupt this process. Screening efforts have identified a class of triphenyl compounds that are structurally distinct from the homoserine lactone autoinducer, yet interact specifically and potently with LasR receptor to modulate quorum sensing (Muh et al., 2006a). Here we present the high-resolution crystal structures of the ligand-binding domain of LasR in complex with the autoinducer N-3-oxo-dodecanoyl homoserine lactone (1.4 Å resolution), and with the triphenyl mimics TP-1, TP-3, and TP-4 (to between 1.8-2.3 Å resolution). These crystal structures provide a molecular rationale for understanding how chemically distinct compounds can be accommodated by a highly selective receptor and provides the framework for the development of novel quorum sensing regulators, utilizing the triphenyl scaffold.

Aging is a biological process characterized by time-dependent functional declines that are influenced by changes in redox status and by oxidative stress-induced inflammatory reactions. An organism's pro-inflammatory status may underlie the aging process and age-related diseases. In this review, we explore the molecular basis of low-grade, unresolved, subclinical inflammation as a major risk factor for exacerbating the aging process and age-related diseases. We focus on the redox-sensitive transcription factors, NF-κB and FOXO, which play essential roles in the expression of pro-inflammatory mediators and anti-oxidant enzymes, respectively. Major players in molecular inflammation are discussed with respect to the age-related up-regulation of pro-inflammatory cytokines and adhesion molecules, cyclo-oxygenase-2, lipoxygenase, and inducible nitric oxide synthase. The molecular inflammation hypothesis proposed by our laboratory is briefly described to give further molecular insights into the intricate interplay among redox balance, pro-inflammatory gene activation, and chronic age-related inflammatory diseases. The final section discusses calorie restriction as an aging-retarding intervention that also exhibits extraordinarily effective anti-inflammatory activity by modulating GSH redox, NF-κB, SIRT1, PPARs, and FOXOs.