Obesity is the most common nutritional disorder in Western society. Uncoupling protein-2 (UCP2) is a recently identified member of the mitochondrial transporter superfamily that is expressed in many tissues, including adipose tissue. Like its close relatives UCP1 and UCP3, UCP2 uncouples proton entry in the mitochondrial matrix from ATP synthesis and is therefore a candidate gene for obesity. We show here that a common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo and results in increased transcription of a reporter gene in the human adipocyte cell line PAZ-6. In analyzing 340 obese and 256 never-obese middle-aged subjects, we found a modest but significant reduction in obesity prevalence associated with the less-common allele. We confirmed this association in a population-based sample of 791 middle-aged subjects from the same geographic area. Despite its modest effect, but because of its high frequency (approximately 63%), the more-common risk allele conferred a relatively large population-attributable risk accounting for 15% of the obesity in the population studied.

Angiotensin II type 2 (AT2) receptors are involved in the inhibition of cell proliferation as well as in apoptosis and neuronal differentiation, through intracellular signalling pathways that remain poorly defined. The present study examines the effect of AT2-receptor stimulation on growth-factor-induced pathways leading to the activation of mitogen-activated protein (MAP) kinases. In N1E-115 neuroblastoma cells, AT2 receptors inhibit the activity of MAP kinases induced by serum as well as by epidermal growth factor. The inhibitory effect of angiotensin II (Ang II) is rapid and transient, and affects both ERK1 and ERK2 (extracellular signal-related protein kinase) isoforms of the enzyme. AT2-mediated MAP kinase inactivation is not sensitive to pertussis toxin or okadaic acid, but involves a vanadate-sensitive protein tyrosine phosphatase (PTP). Expression of MAP kinase phosphatase-1 (MKP-1) is not significantly modified upon AT2-receptor activation, and insensitivity to actinomycin D also rules out transcriptional induction of other MKPs as a possible mechanism for AT2-mediated inactivation of MAP kinases. In addition, we report here that both in N1E-115 cells and in Chinese hamster ovary cells expressing recombinant human AT2 receptors, Ang II rapidly stimulates the catalytic activity of SHP-1, a soluble PTP that has been implicated in termination of signalling by cytokine and growth-factor receptors. These findings thus demonstrate functional negative cross-talk between heptahelical AT2 receptors and receptor tyrosine kinases, and suggest that SHP-1 tyrosine phosphatase is an early transducer of the AT2 receptor signalling pathway.

Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

The presence and properties of serum autoantibodies against fi-adrenergic receptors in patients with idiopathic dilated cardiomyopathy were studied using synthetic peptides derived from the predicted sequences of the human fi-adrenergic receptors.Peptides corresponding to the sequences of the second extracellular loop of the human A,-and t92-adrenergic receptors were used as antigens in an enzyme immunoassay to screen sera from patients with dilated cardiomyopathy (n = 42), ischemic heart disease (n = 17), or healthy blood donors (n = 34). The sera of thirteen dilated cardiomyopathy patients, none of the ischemic heart disease patients, and four of the healthy controls monospecifically recognized the jfl-peptide. Only affinity-purified antibodies of these patients had a inhibitory effect on radioligand binding to the fl, receptor of C6 rat glioma cells. They recognized the receptor protein by immunoblot and bound in situ to human myocardial tissue. We conclude that a subgroup of patients with idiopathic dilated cardiomyopathy have in their sera autoantibodies specifically directed against the second extracellular loop of the fI-adrenergic receptor. These antibodies could serve as a marker of an autoimmune response with physiological and/or pathological implications. (J.

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The structural properties of a number of proteins belonging to the family of receptors coupled to GTP binding proteins are discussed in relation to their function.The structure of the ligand binding site and of the regions involved in coupling to the G proteins are analyzed mainly for the adrenergic and muscarinic cholinergic receptors, for which site-directed mutagenesis and chimaeric constructions have been studied. The structure of the genes are compared and the presence of various regulatory elements is discussed in relation to control of expression.Mechanism of desensitization and internalization, while mostly studied for the j2-adrenergic receptor, are proposed to be generally applicable to all G-protein-coupled receptors.A wide variety of membrane receptors for hormones and neurotransmitters are coupled to guanine-nucleotide-binding regulatory 'G' proteins, which upon activation by receptors, stimulate or inhibit various effectors such as enzymes or ion channels. Among the family of receptors coupled to G proteins are those for adrenaline and other catecholamines, the 'adrenergic receptors' and those for acetylcholine and related muscarinic ligands, the 'muscarinic cholinergic' receptors. Other similar proteins belonging to this growing family are those for serotonine, for dopamine, for the tachykinins, and for the pituitary glycoprotein hormones, to mention but a few (Table 1).Several subtypes of these receptors have been defined on the basis of pharmacologic properties and more have been found recently through cloning of homologous genes. Expression of individual receptor subtypes in cells initially devoid of receptors reveal characteristic ligand-binding profiles and activation of different intracellular effector systems. The p1-, j2-and j3-adrenergic, the 5HT-1A serotoninergic (5HT = 5-hydroxytryptamine) and the D1 dopaminergic receptors through activation of a G, (GTP binding stimulatory) protein stimulate the membrane-bound adenylyl cyclase, leading to an increased production of cyclic AMP. The a2A-and a2B- Abbreviations. PAR, /I-adrenergic receptor; E-R7G, family of receptors having seven transmembrane domains, coupled to GTP binding proteins and having an extra long (= 333 residues) extracellular domain; G proteins, GTP binding regulatory proteins (G, = inhibitory, G, = stimulatory, G, = coupled to phospholipase); GRE, glucocorticoid responsive elements; 5HT, 5-hydroxytryptamine; m, muscarinic; R7G, family of receptors having seven transmembrane domains and coupled to GTP binding proteins. adrenergic, the m2 and m4 muscarinic cholinergic, the D2 dopaminergic receptors, through activation of a Gi (GTP binding inhibitory) protein, inhibit this cyclase, whereas the El-adrenergic, the m l , m3 and m5 muscarinic and the 5HT-1C and 5HT-2 serotoninergic receptors through coupling either to a Gi or a Go (GTP binding, coupled to other effectors) protein modulate the hydrolysis of polyphosphoinositol lipids by phospholipase A2 or C leading to the production of two second messengers, inositol tri(tetra)phosp...

The peroxisome proliferator-activated receptors (PPAR) ␣ and ␥ play key roles in the transcriptional control of contrasting metabolic pathways such as adipogenesis and fatty acid ␤-oxidation. Both ligand-activated nuclear receptors bind to common target gene response elements and interact with distinct domains of the transcriptional coactivator PGC-1 to attain their full transcriptional potency. Thus, PPAR subtype specificity may be determined by ligand availability and transcription factor or coactivator expression levels. To identify other, perhaps more precise mechanisms contributing to PPAR subtype specificity, we studied PGC-1 recruitment by PPARs using a previously described hormone response element in the human UCP1 promoter and a human brown adipocyte cell line as our model system. As in rodents, PGC-1 is involved in the transcriptional regulation of the UCP1 gene in humans and mediates the effects of PPAR␣ and PPAR␥ agonists and retinoic acid. Interestingly, a previously postulated PGC-1 repressor selectively affects the PPAR␣-mediated activation of UCP1 gene expression. Furthermore, inhibition of p38 MAPK signaling, known to regulate the PGC-1/ repressor interaction, decreases the stimulatory effect of PPAR␣ agonist treatment without reducing the response to thiazolidinedione or retinoic acid. These data support a model whereby PPAR subtype specificity is regulated by recruitment of PGC-1. Transcription of the uncoupling protein-1 (UCP1)1 gene is almost exclusively restricted to brown adipose tissue (BAT) where its major function in diet-induced and in non-shivering thermogenesis depends on the capacity to uncouple oxidative phosphorylation from ATP generation (1). Several physiological stimuli including cold exposure and sympathetic signaling via norepinephrine and cAMP as well as thyroid hormone regulate UCP1 gene expression in rodents (2-5). In addition, retinoids (6, 7) and synthetic ligands for the peroxisome proliferator-activated receptors PPAR␣ (8) and PPAR␥ (9, 10) increase rodent UCP1 gene expression. A multipartite response element with partial homology to rat and mouse UCP1 enhancers has been identified recently (11) in humans. Located within this element is a putative binding site for cAMP-response elementbinding protein mediating the effect of catecholamines on UCP1 gene expression as well as a complex element containing consensus recognition sites for the retinoic acid receptors, PPAR␣ and PPAR␥.The PPARs are ligand-activated transcription factors and members of the nuclear hormone receptor superfamily (12). PPAR␥ is a master regulator of adipocyte differentiation (13), as evidenced by induction of adipose tissue markers in fibroblasts and myoblasts upon its ectopic overexpression (14), and controls the expression of genes involved in fatty acid synthesis and glucose metabolism (reviewed in Ref. 15). PPAR␣ is involved in fatty acid catabolism by regulating the expression of genes encoding fatty acid oxidation enzymes (12,16,17). As in some other tissues, PPAR␣ and PPAR␥ are both expresse...