Thomas P. Sakmar

Defeating Alzheimer's disease and other dementias: a priority for European science and society

Defeating Alzheimers disease and other dementias : a priority for European science and society

Structural Determinants of the Supramolecular Organization of G Protein-Coupled Receptors in Bilayers

The G protein-coupled receptor (GPCR) rhodopsin self-assembles into supramolecular structures in native bilayers, but the structural determinants of receptor oligomerization are not known. We carried out multiple self-assembly coarse-grained molecular dynamics (CGMD) simulations of model membranes containing up to 64 molecules of the visual receptor rhodopsin over time scales reaching 100 μs. The simulations show strong preferential interaction modes between receptors. Two primary modes of receptor-receptor interactions are consistent with umbrella sampling/potential of mean force (PMF) calculations as a function of the distance between a pair of receptors. The preferential interfaces, involving helices (H) 1/8, 4/5 and 5, present no energy barrier to forming a very stable receptor dimer. Most notably, the PMFs show that the preferred rhodopsin dimer exists in a tail-to-tail conformation, with the interface comprising transmembrane H1/H2 and amphipathic H8 at the extracellular and cytoplasmic surfaces, respectively. This dimer orientation is in line with earlier electron microscopy, X-ray, and cross-linking experiments of rhodopsin and other GPCRs. Less stable interfaces, involving H4 and H6, have a free energy barrier for desolvation (delipidation) of the interfaces and appear to be designed to stabilize lubricated (i.e., lipid-coated) dimers. The overall CGMD strategy used here is general and can be applied to study the homo- and heterodimerization of GPCRs and other transmembrane proteins. Systematic extension of the work will deepen our understanding of the forces involved in the membrane organization of integral membrane proteins.

Update on Alzheimer's Disease Therapy and Prevention Strategies

Alzheimers disease (AD) is the primary cause of age-related dementia. Effective strategies to prevent and treat AD remain elusive despite major efforts to understand its basic biology and clinical pathophysiology. Significant investments in therapeutic drug discovery programs over the past two decades have yielded some important insights but no blockbuster drugs to alter the course of disease. Because significant memory loss and cognitive decline are associated with neuron death and loss of gray matter, especially in the frontal cortex and hippocampus, some focus in drug development has shifted to early prevention of cellular pathology. Although clinical trial design is challenging, due in part to a lack of robust biomarkers with predictive value, some optimism has come from the identification and study of inherited forms of early-onset AD and genetic risk factors that provide insights about molecular pathophysiology and potential drug targets. In addition, better understanding of the Aβ amyloid pathway and the tau pathway-leading to amyloid plaques and neurofibrillary tangles, respectively, which are histopathological hallmarks of AD-continues to drive significant drug research and development programs. The main focus of this review is to summarize the most recent basic biology, biochemistry, and pharmacology that serve as a foundation for more than 50 active advanced-phase clinical trials for AD prevention and therapy.

Rhodopsin Forms a Dimer with Cytoplasmic Helix 8 Contacts in Native Membranes

G protein-coupled receptors form dimers and higher-order oligomers in membranes, but the precise mode of receptor-receptor interaction remains unknown. To probe the intradimeric proximity of helix 8 (H8), we conducted chemical cross-linking of endogenous cysteines in rhodopsin in disk membranes. We identified a Cys316-Cys316 cross-link using partial proteolysis and liquid chromatography with mass spectrometry. These results show that a symmetric dimer interface mediated by H1 and H8 contacts is present in native membranes.

Structural evidence for a sequential release mechanism for activation of heterotrimeric g proteins.

Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5-diphosphate (GDP) release by the G protein alpha-subunit is not well understood. Amino acid substitutions in the conserved alpha5 helix of G(i), which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Galpha(i1)-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Galpha(i1)-T329A.GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg(+2) coordination sphere and dislodge bound Mg(+2). We propose a sequential release mechanism whereby a transient conformational change in the alpha5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.

Insider access: pepducin symposium explores a new approach to GPCR modulation

The inaugural Pepducin Science Symposium convened in Cambridge, Massachusetts on March 8–9, 2009 provided the opportunity for an international group of distinguished scientists to present and discuss research regarding G protein–coupled receptor‐related research. G protein–coupled receptors (GPCRs) are, arguably, one of the most important molecular targets in drug discovery and pharmaceutical development today. This superfamily of membrane receptors is central to nearly every signaling pathway in the human body and has been the focus of intense research for decades. However, as scientists discover additional properties of GPCRs, it has become clear that much is yet to be understood about how these receptors function. Everyone agrees, however, that tremendous potential remains if specific GPCR signaling pathways can be modulated to correct pathological states. One exciting new approach to this challenge involves pepducins: novel, synthetic lipopeptide pharmacophores that modulate heptahelical GPCR activity from inside the cell membrane.

Mapping Substance P Binding Sites on the Neurokinin-1 Receptor Using Genetic Incorporation of a Photoreactive Amino Acid

Background: Unnatural amino acids can be genetically incorporated into 7-transmembrane receptors. Results: A photoreactive amino acid introduced into the neurokinin-1 receptor cross-links substance P to the N-terminal and extracellular loop II domains of the receptor. Conclusion: The extracellular domain of the neurokinin-1 receptor possesses multiple potential binding sites for substance P. Significance: A photocross-linking methodology reveals novel interaction sites in the neurokinin-1-receptor-substance P complex. Substance P (SP) is a neuropeptide that mediates numerous physiological responses, including transmission of pain and inflammation through the neurokinin-1 (NK1) receptor, a G protein-coupled receptor. Previous mutagenesis studies and photoaffinity labeling using ligand analogues suggested that the binding site for SP includes multiple domains in the N-terminal (Nt) segment and the second extracellular loop (ECLII) of NK1. To map precisely the NK1 residues that interact with SP, we applied a novel receptor-based targeted photocross-linking approach. We used amber codon suppression to introduce the photoreactive unnatural amino acid p-benzoyl-l-phenylalanine (BzF) at 11 selected individual positions in the Nt tail (residues 11–21) and 23 positions in the ECLII (residues 170(C-10)–193(C+13)) of NK1. The 34 NK1 variants were expressed in mammalian HEK293 cells and retained the ability to interact with a fluorescently labeled SP analog. Notably, 10 of the receptor variants with BzF in the Nt tail and 4 of those with BzF in ECLII cross-linked efficiently to SP, indicating that these 14 sites are juxtaposed to SP in the ligand-bound receptor. These results show that two distinct regions of the NK1 receptor possess multiple determinants for SP binding and demonstrate the utility of genetically encoded photocross-linking to map complex multitopic binding sites on G protein-coupled receptors in a cell-based assay format.

Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors.

Novel methods are required for site‐specific, quantitative fluorescent labeling of G‐protein‐coupled receptors (GPCRs) and other difficult‐to‐express membrane proteins. Ideally, fluorescent probes should perturb the native structure and function as little as possible. We evaluated bioorthogonal reactions to label genetically encoded p‐acetyl‐L‐phenylalanine (AcF) or p‐azido‐L‐phenylalanine (azF) residues in receptors heterologously expressed in mammalian cells. We found that keto‐selective reagents were not truly bioorthogonal, possibly owing to post‐translational protein oxidation reactions. In contrast, the strain‐promoted [3+2] azide–alkyne cycloaddition (SpAAC) with dibenzocyclooctyne (DIBO) reagents yielded stoichiometric conjugates with azF‐rhodopsin while undergoing negligible background reactions. As one application of this technique, we used Alexa488–rhodopsin to measure the kinetics of ligand uptake and release in membrane‐mimetic bicelles using a novel fluorescence‐quenching assay.

Unnatural Amino Acid Mutagenesis of GPCRs Using Amber Codon Suppression and Bioorthogonal Labeling

To advance dynamic, temporal, and kinetic studies of the G protein-coupled receptor (GPCR) signalosome, new approaches are required to introduce non- or minimally perturbing labels or probes into expressed receptors. We report here a series of methods that are based on unnatural amino acid mutagenesis of GPCRs using amber codon suppression technology. We show that labeling reactions at genetically introduced keto moieties (p-acetyl-L-Phe/AcF and p-benzoyl-L-Phe/BzF) are not completely bioorthogonal due to protein oxidation, which causes high background. However, labeling reactions that target p-azido-L-Phe (azF) using the Staudinger-Bertozzi ligation and the strain-promoted alkyne-azide cycloaddition are bioorthogonal and are satisfactory for introducing labels or probes at near quantitative efficiency under mild labeling conditions. To our knowledge, this is the first report of a site-specific modification of an azF residue with a dibenzocyclooctyne-derivatized fluorophore. The methodologies we discuss are general, in that they can be applied in principle to any amino acid position in any expressed GPCR.

CXC Chemokine Receptor 3 Alternative Splice Variants Selectively Activate Different Signaling Pathways

The G protein-coupled receptor (GPCR) C-X-C chemokine receptor 3 (CXCR3) is a potential drug target that mediates signaling involved in cancer metastasis and inflammatory diseases. The CXCR3 primary transcript has three potential alternative splice variants and cell-type specific expression results in receptor variants that are believed to have different functional characteristics. However, the molecular pharmacology of ligand binding to CXCR3 alternative splice variants and their downstream signaling pathways remain poorly explored. To better understand the role of the functional consequences of alternative splicing of CXCR3, we measured signaling in response to four different chemokine ligands (CXCL4, CXCL9, CXCL10, and CXCL11) with agonist activity at CXCR3. Both CXCL10 and CXCL11 activated splice variant CXCR3A. Whereas CXCL10 displayed full agonistic activity for Gαi activation and extracellular signal regulated kinase (ERK) 1/2 phosphorylation and partial agonist activity for β-arrestin recruitment, CXCL9 triggered only modest ERK1/2 phosphorylation. CXCL11 induced CXCR3B-mediated β-arrestin recruitment and little ERK phosphorylation. CXCR3Alt signaling was limited to modest ligand-induced receptor internalization and ERK1/2 phosphorylation in response to chemokines CXCL11, CXCL10, and CXCL9. These results show that CXCR3 splice variants activate different signaling pathways and that CXCR3 variant function is not redundant, suggesting a mechanism for tissue specific biased agonism. Our data show an additional layer of complexity for chemokine receptor signaling that might be exploited to target specific CXCR3 splice variants.