Nongenomic response pathways mediate many of the rapid actions of steroid hormones, but the mechanisms underlying such responses remain controversial. In some cases, cell-surface expression of classical nuclear steroid receptors has been suggested to mediate these effects, but, in a few instances, specific G-protein-coupled receptors (GPCRs) have been reported to be responsible. Here, we describe the activation of a novel, neuronally expressed Drosophila GPCR by the insect ecdysteroids ecdysone (E) and 20-hydroxyecdysone (20E). This is the first report of an identified insect GPCR interacting with steroids. The Drosophila melanogaster dopamine/ecdysteroid receptor (DmDopEcR) shows sequence homology with vertebrate ␤-adrenergic receptors and is activated by dopamine (DA) to increase cAMP levels and to activate the phosphoinositide 3-kinase pathway. Conversely, E and 20E show high affinity for the receptor in binding studies and can inhibit the effects of DA, as well as coupling the receptor to a rapid activation of the mitogen-activated protein kinase pathway. The receptor may thus represent the Drosophila homolog of the vertebrate "␥-adrenergic receptors," which are responsible for the modulation of various activities in brain, blood vessels, and pancreas. Thus, DmDopEcR can function as a cell-surface GPCR that may be responsible for some of the rapid, nongenomic actions of ecdysteroids, during both development and signaling in the mature adult nervous system.

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A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (NPFR76F, also called GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62-66% similarity and 32-34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein Galpha16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila.

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1 The e ects of substitution of the Ser 200 and Ser 204 residues with alanine on the signalling properties of the cloned human a 2A -adrenoceptor, stably expressed in Chinese hamster ovary (CHO) cell lines, have been investigated using noradrenaline and the structural isomers of octopamine. 2 The Ser?Ala 200 or the Ser?Ala 204 mutant forms of the a 2A -adrenoceptor, when expressed in cells in the absence of pertussis toxin pretreatment, are two orders of magnitude more sensitive to inhibition of cyclic AMP production by (+)-para-octopamine and (+)-meta-octopamine, respectively, than cells expressing the wild-type receptor. Binding studies indicate that the e ects are not due to an increased agonist a nity for the mutant receptors and that they are likely to be due to agonist-mediated conformational changes in receptor structure. 3 After incubation with pertussis toxin, (+)-meta-octopamine (100 mM and above) produced a stimulation of cyclic AMP levels in cells expressing the Ser?Ala 204 mutant form of the a 2A -adrenoceptor but showed no stimulation in cells expressing the Ser?Ala 200 mutant receptor. Under these conditions (+)-para-octopamine did not produce any increases in cyclic AMP production in cells expressing either of the mutant receptor forms or the wild-type receptor. 4 The results emphasise the importance of the Ser 200 and Ser 204 residues of the a 2A -adrenoceptor in exerting an inhibitory in¯uence on the ability of (+)-para-octopamine and (+)-meta-octopamine respectively, to induce a receptor-agonist conformation capable of inhibiting forskolin-stimulation of cyclic AMP levels. 5 It is clear that Ser 204 also prevents meta-octopamine from generating a receptor-agonist conformation that can increase cyclic AMP levels, emphasising the importance of this residue in the agonist-speci®c coupling of this receptor to di erent second messenger systems.

Using recombinant cinnamyl alcohol dehydrogenase isoform 2 (CAD2, EC 1.1.1.195), an NADPH-dependent aromatic alcohol dehydrogenase involved in lignification in vascular plants, we have investigated the detailed steady-state kinetic mechanism of CAD2 and the role of a serine residue in determining the cofactor specificity of CAD2. Site-directed mutagenesis (S212D) and overexpression of the WT and mutant S212D forms of CAD2 in Escherichia coli, followed by kinetic studies on the purified WT and mutant proteins, confirmed the involvement of S212D in recognizing the phosphate group of NADPH and provided information on the structural requirements for NADPH specificity. From substrate kinetic patterns and product inhibition studies both WT and S212D mutant forms of CAD2 have been shown to follow rapid equilibrium random bireactant kinetics with the value of the interaction factor (alpha) for WT (0.25) being significantly less than that for S212D CAD2 (0.45). The changes in binding energy arising from the mutation on the binding of the 2'-phosphate site of the coenzyme were assessed. A marked degree of physical interaction was detected between the enzymatic binding sites of the coniferyl alcohol substrate and the 2'-phosphate binding region, which are quite distant in the three-dimensional structure. The inhibition by 2',5'-ADP and 5'-AMP was found to be weak for both WT and S212D CAD2. Strong substrate inhibition was detected for CAD2, and its implications for plant physiological studies were assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of cholecystokinin (CCK) and gastrin (G) receptors in human gastrointestinal cancers remains poorly documented and is still of a controversial nature. We have measured the levels of mRNA for CCK‐A and CCK‐B/gastrin receptors using quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) in primary digestive cancers and hepatic metastases. CCK‐A‐receptor mRNA was detected in 5 out of 8 esophageal cancers (0.1–1 fg/μg), in 5 out of 8 gastric cancers (0.05–4.2 fg/μg) and in 5 out of 12 colon cancers (0.1–1 fg/μg RNA). CCK‐B/gastrin mRNA was not detected in esophageal cancers but was detected in 7 out of 8 gastric cancers (0.05–5.2 fg/μg), and in only 2 out of 12 colon adenocarcinomas (0.05–1 fg/μg RNA). The expression of the CCK‐A receptor in esophageal, gastric and colon cancers and of the CCK‐B/gastrin receptor in the majority of gastric adenocarcinomas screened may be an important indicator of the influence of CCK and gastrin of local or systemic origin on the growth of these cancers. Int. J. Cancer 72:931–936, 1997. © 1997 Wiley‐Liss, Inc.

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