Heptahelical receptors communicate extracellular information to the cytosolic compartment by binding an extensive variety of ligands. They do so through conformational changes that propagate to intracellular signaling partners as the receptor switches from a resting to an active conformation. This active state has been classically considered unique and responsible for regulation of all signaling pathways controlled by a receptor. However, recent functional studies have challenged this notion and called for a paradigm where receptors would exist in more than one signaling conformation. This study used bioluminescence resonance energy transfer assays in combination with ligands of different functional profiles to provide in vivo physical evidence of conformational diversity of ␦-opioid receptors (DORs). DORs and ␣ i1 ␤ 1 ␥ 2 G protein subunits were tagged with Luc or green fluorescent protein to produce bioluminescence resonance energy transfer pairs that allowed monitoring DOR-G protein interactions from different vantage points. Results showed that DORs and heterotrimeric G proteins formed a constitutive complex that underwent structural reorganization upon ligand binding. Conformational rearrangements could not be explained by a two-state model, supporting the idea that DORs adopt ligand-specific conformations. In addition, conformational diversity encoded by the receptor was conveyed to the interaction among heterotrimeric subunits. The existence of multiple active receptor states has implications for the way we conceive specificity of signal transduction.Heptahelical receptors are versatile membrane proteins that play an important role in cellular communication. They do so by recognizing a large variety of extracellular ligands that convey information to the intracellular compartment by modifying receptor conformation upon binding. These structural modifications then trigger an array of biochemical changes that ultimately control vital processes within the cell. Traditionally, conformational changes induced by ligand binding have been thought to shift equilibrium between an active and an inactive conformation of the receptor. According to this classical view, all agonists would stabilize a single active state that equally effectively stimulates all signaling pathways controlled by the receptor (1). In consequence, ligand ability to stabilize this unique active state would be the only determinant of ligand efficacy at all functional readouts. Furthermore, this model predicts that ligand rank order of efficacy across different readouts should be maintained, representing a progressive accumulation (or decrease) of the single active state of the receptor (2).However, recent data have challenged this view, suggesting that the complexity of heptahelical receptor signaling cannot be based on the accumulation of a single active receptor conformation, and efficacy cannot be restricted to only a quantitative dimension (3, 4). Evidence supporting this assertion has been largely based on functional studies whose results ...

Anticancer treatments for childhood acute lymphoblastic leukaemia (ALL) are highly effective but are now implicated in causing impaired muscle function in long-term survivors. However, no comprehensive assessment of skeletal muscle mitochondrial functions in long-term survivors has been performed and the presence of persistent chemotherapy-induced skeletal muscle mitochondrial dysfunction remains a strong possibility. Non-tumour-bearing mice were treated with two drugs that have been used frequently in ALL treatment (doxorubicin and dexamethasone) for up to 4 cycles at 3-week intervals and euthanized 3 months after the 4th cycle. Treated animals had impaired growth and lower muscle mass as well as reduced mitochondrial respiration and increased reactive oxygen species production per unit oxygen consumption. Mitochondrial DNA content and protein levels of key mitochondrial membrane proteins and markers of mitochondrial biogenesis were unchanged, but protein levels of Parkin were reduced. This suggests a novel pattern of chemotherapy-induced mitochondrial dysfunction in skeletal muscle that persists because of an acquired defect in mitophagy signaling. The results could explain the observed functional impairments in adult survivors of childhood ALL and may also be relevant to long-term survivors of other cancers treated with similar regimes.

Phosphatidylinositol 3-kinase (PI3K) regulates many cellular functions including growth and survival, and its excessive activation is a hallmark of cancer. Somatostatin, acting through its G protein-coupled receptor (GPCR) sst2, has potent proapoptotic and anti-invasive activities on normal and cancer cells. Here, we report a novel mechanism for inhibiting PI3K activity. Somatostatin, acting through sst2, inhibits PI3K activity by disrupting a preexisting complex comprising the sst2 receptor and the p85 PI3K regulatory subunit. Surface plasmon resonance and molecular modeling identified the phosphorylated-Y 71 residue of a p85-binding pYXXM motif in the first sst2 intracellular loop, and p85 COOH-terminal SH2 as direct interacting domains. Somatostatin-mediated dissociation of this complex as well as p85 tyrosine dephosphorylation correlates with sst2 tyrosine dephosphorylation on the Y 71 residue. Mutating sst2-Y 71 disabled sst2 to interact with p85 and somatostatin to inhibit PI3K, consequently abrogating sst2's ability to suppress cell survival and tumor growth. These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand-activated GPCR of PI3K-dependent survival pathways, which may be an important molecular target for antineoplastic therapy.

The Cholecystokinin-1 receptor (CCK1R) mediates actions of CCK in areas of the central nervous system and of the gut. It is a potential target to treat a number of diseases. As for all G-protein-coupled receptors, docking of ligands into modeled CCK1R binding site should greatly help to understand intrinsic mechanisms of activation. Here, we describe the procedure we used to progressively build a structural model for the CCK1R, to integrated, and on the basis of site-directed mutagenesis data on its binding site. Reliability of the CCK1R model was confirmed by interaction networks that involved conserved and functionally crucial motifs in G-protein-coupled receptors, such as Glu/Asp-Arg-Tyr and Asn-Pro-Xaa-Xaa-Tyr motifs. In addition, the 3-D structure of CCK1R-bound CCK resembled that determined by NMR in a lipid environment. The derived computational model was also used for revealing binding modes of several nonpeptide ligands and for rationalizing ligand structure-activity relationships known from experiments. Our findings indeed support that our "validated CCK1R model" could be used to study the intrinsic mechanism of CCK1R activation and design new ligands.