Adiponectin (also known as 30-kDa adipocyte complement-related protein; Acrp30) is a hormone secreted by adipocytes that acts as an antidiabetic and anti-atherogenic adipokine. Levels of adiponectin in the blood are decreased under conditions of obesity, insulin resistance and type 2 diabetes. Administration of adiponectin causes glucose-lowering effects and ameliorates insulin resistance in mice. Conversely, adiponectin-deficient mice exhibit insulin resistance and diabetes. This insulin-sensitizing effect of adiponectin seems to be mediated by an increase in fatty-acid oxidation through activation of AMP kinase and PPAR-alpha. Here we report the cloning of complementary DNAs encoding adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) by expression cloning. AdipoR1 is abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver. These two adiponectin receptors are predicted to contain seven transmembrane domains, but to be structurally and functionally distinct from G-protein-coupled receptors. Expression of AdipoR1/R2 or suppression of AdipoR1/R2 expression by small-interfering RNA supports our conclusion that they serve as receptors for globular and full-length adiponectin, and that they mediate increased AMP kinase and PPAR-alpha ligand activities, as well as fatty-acid oxidation and glucose uptake by adiponectin.

Cloning of adiponectin receptors that mediate antidiabetic metabolic effects

The neurohypophysial peptide oxytocin (OT) and OT-like hormones facilitate reproduction in all vertebrates at several levels. The major site of OT gene expression is the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. In response to a variety of stimuli such as suckling, parturition, or certain kinds of stress, the processed OT peptide is released from the posterior pituitary into the systemic circulation. Such stimuli also lead to an intranuclear release of OT. Moreover, oxytocinergic neurons display widespread projections throughout the central nervous system. However, OT is also synthesized in peripheral tissues, e.g., uterus, placenta, amnion, corpus luteum, testis, and heart. The OT receptor is a typical class I G protein-coupled receptor that is primarily coupled via Gq proteins to phospholipase C-β. The high-affinity receptor state requires both Mg2+ and cholesterol, which probably function as allosteric modulators. The agonist-binding region of the receptor has bee...

The Oxytocin Receptor System: Structure, Function, and Regulation

Natural products: a continuing source of novel drug leads.

Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 A/3.7 A resolution. The cytoplasmic ends of the beta2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the beta2AR are not seen. The beta2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the beta2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.

Crystal structure of the human beta2 adrenergic G-protein-coupled receptor.

Among membrane‐bound receptors, the G protein‐coupled receptors (GPCRs) are certainly the most diverse. They have been very successful during evolution, being capable of transducing messages as different as photons, organic odorants, nucleotides, nucleosides, peptides, lipids and proteins. Indirect studies, as well as two‐dimensional crystallization of rhodopsin, have led to a useful model of a common ‘central core’, composed of seven transmembrane helical domains, and its structural modifications during activation. There are at least six families of GPCRs showing no sequence similarity. They use an amazing number of different domains both to bind their ligands and to activate G proteins. The fine‐tuning of their coupling to G proteins is regulated by splicing, RNA editing and phosphorylation. Some GPCRs have been found to form either homo‐ or heterodimers with a structurally different GPCR, but also with membrane‐bound proteins having one transmembrane domain such as nina‐A, odr‐4 or RAMP, the latter being involved in their targeting, function and pharmacology. Finally, some GPCRs are unfaithful to G proteins and interact directly, via their C‐terminal domain, with proteins containing PDZ and Enabled/VASP homology (EVH)‐like domains.

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Molecular tinkering of G protein‐coupled receptors: an evolutionary success

Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors.

Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

Traditional screening paradigms often focus on single targets. To facilitate drug discovery in the more complex physiological environment of a cell or organism, powerful cellular imaging systems have been developed. The emergence of these detection technologies allows the quantitative analysis of cellular events and visualization of relevant cellular phenotypes. Cellular imaging facilitates the integration of complex biology into the screening process, and addresses both high-content and high-throughput needs. This review describes how cellular imaging technologies contribute to the drug discovery process.

Cellular imaging in drug discovery

In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E/DRY sequence, in the activation process of the α1B-adrenergic receptor (α1B-AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural/dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic/hydrophilic character of D142, which could be regulated by protonation/deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the α1B-AR. Our study represents an example of quantitative structure-activity relationship analysis of the activation process of a G protein-coupled receptor.

The activation process of the α1B-adrenergic receptor: Potential role of protonation and hydrophobicity of a highly conserved aspartate

Mutations of G protein-coupled receptors can increase their constitutive (agonist-independent) activity Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases The analysis of the constitutively active G protein-coupled receptors has provided important informations about the molecular mechanisms underlying receptor activation and drug action

Constitutively active G protein-coupled receptors: potential mechanisms of receptor activation.

We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the α1b-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the α1b-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the α1b-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type α1b-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the α1b-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.

Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation.

Alexander Scheer

Papers

Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

Cellular imaging in drug discovery

The activation process of the α1B-adrenergic receptor: Potential role of protonation and hydrophobicity of a highly conserved aspartate

Constitutively active G protein-coupled receptors: potential mechanisms of receptor activation.

Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation.