Thin-section CT findings of peripheral lung adenocarcinomas correlate well with histologic prognostic factors.

Transmembrane signal transduction via heterotrimeric G proteins is reported to be inhibited by RGS (regulators of G-protein signalling) proteins. These RGS proteins work by increasing the GTPase activity of G protein alpha-subunits (G alpha), thereby driving G proteins into their inactive GDP-bound form. However, it is not known how RGS proteins regulate the kinetics of physiological responses that depend on G proteins. Here we report the isolation of a full-length complementary DNA encoding a neural-tissue-specific RGS protein, RGS8, and the determination of its function. We show that RGS8 binds preferentially to the alpha-subunits G(alpha)o and G(alpha)i3 and that it functions as a GTPase-activating protein (GAP). When co-expressed in Xenopus oocytes with a G-protein-coupled receptor and a G-protein-coupled inwardly rectifying K+ channel (GIRK1/2), RGS8 accelerated not only the turning off but also the turning on of the GIRK1/2 current upon receptor stimulation, without affecting the dose-response relationship. We conclude that RGS8 accelerates the modulation of G-protein-coupled channels and is not just a simple negative regulator. This property of RGS8 may be crucial for the rapid regulation of neuronal excitability upon stimulation of G-protein-coupled receptors.

Adenosine and its endogenous precursor ATP are main components of the purinergic system that modulates cellular and tissue functions via specific adenosine and ATP receptors (P1 and P2 receptors), respectively. Although adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the ability of P1 and P2 receptors to form new functional structures such as a heteromer to control the complex purinergic cascade. Here we have shown that G i/o protein-coupled A1 adenosine receptor (A1R) and Gq proteincoupled P2Y1 receptor (P2Y1R) coimmunoprecipitate in cotransfected HEK293T cells, suggesting the oligomeric association between distinct G protein-coupled P1 and P2 receptors. A 1R and P2Y2 receptor, but not A1R and dopamine D2 receptor, also were found to coimmunoprecipitate in cotransfected cells. A 1R agonist and antagonist binding to cell membranes were reduced by coexpression of A 1R and P2Y1R, whereas a potent P2Y1R agonist adenosine 5-O-(2-thiotriphosphate) (ADP␤S) revealed a significant potency to A1R binding only in the cotransfected cell membranes. Moreover, the A 1R͞P2Y1R coexpressed cells showed an ADP␤S-dependent reduction of forskolin-evoked cAMP accumulation that was sensitive to pertussis toxin and A1R antagonist, indicating that ADP␤S binds A1R and inhibits adenylyl cyclase activity via Gi/o proteins. Also, a high degree of A1R and P2Y1R colocalization was demonstrated in cotransfected cells by double immunofluorescence experiments with confocal laser microscopy. These results suggest that oligomeric association of A 1R with P2Y1R generates A 1R with P2Y1R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purine signaling.A denosine and ATP are two major neurotransmitter and neuromodulating systems that share a number of structural and functional characteristics. These purinergic systems modulate many physiological processes, including smooth muscle contraction, immune response, platelet aggregation, pain, cardiac function, cardioprotection, and neurotransmission (1). Pharmacological and molecular cloning studies have identified two purinergic receptor families, named adenosine receptor or P1 receptor and ATP receptor or P2 receptor. P1 receptors have been further subdivided into A 1 R, A 2A R, A 2B R, and A 3 R, all of which are G protein-coupled receptors (GPCRs). A 1 and A 3 adenosine receptors are coupled to the inhibition of adenylyl cyclase via G i/o proteins. A 2A and A 2B adenosine receptors are coupled to stimulation of adenylyl cyclase via G s proteins. P2 receptors also are subclassified as P2X or P2Y receptors. To date, seven mammalian P2X receptors (P2X 1-7 R) that are ligand-gated ion channels and five mammalian P2Y receptors (P2Y 1 R , P2Y 2 R , P2Y 4 R , P2Y 6 R, and P2Y 11 R) have been cloned. P2Y receptors are GPCRs that are mainly coupled to phospholipase C via G q proteins. Although the individual pharmacological and biochemical profile of cloned P1 and P2 receptor subtype...

The extracellular domain of the metabotropic glutamate receptor 1alpha (mGluR1alpha) forms a dimer and the ligand, glutamate, induces a structural rearrangement in this domain. However, the conformational change in the cytoplasmic domain, which is critical for mGluR1alpha's interaction with G proteins, remains unclear. Here we investigated the ligand-induced conformational changes in the cytoplasmic domain by fluorescence resonance energy transfer (FRET) analysis of mGluR1alpha labeled with fluorescent protein(s) under total internal reflection field microscopy. Upon ligand binding, the intersubunit FRET efficiency between the second loops increased, whereas that between first loops decreased. In contrast, the intrasubunit FRET did not change clearly. These results show that ligand binding does not change the structure of each subunit, but does change the dimeric allocation of the cytoplasmic regions, which may underlie downstream signaling.

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Tryptophan 5-monooxygenase was purified approximately 5500-fold, to apparent homogeneity with a specific activity of 374 nmol min-' mg-' at 30 "C, from rat brain-stem using Sepharose CL-6B, DEAE-Sepharose CL-6B and pteridine-agarose chromatography. Two distinct active forms were separable by DEAE-Sepharose CL-6B and designated as form I and form I1 based on their order of elution from the gel column. The apparent molecular weight of form I was determined to be 300000 by gel filtration on Ultrogel AcA 34 and 288000 by gradient polyacrylamide gel electrophoresis. The enzyme gave a single band on sodium dodecylsulfate/polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be 59 000, indicating that the enzyme might be composed of four identical subunits. The tetrameric structure of the enzyme was further suggested by crosslinking studies using dimethyl suberimidate as a bifunctional reagent. The enzyme activity was stimulated approximately 3.5-fold by the addition of Fe2+. Kinetic studies revealed that this activation was associated with an increase of V value. The purified enzyme had an activity of phenylakdnine hydroxylation but not an activity of tyrosine hydroxylation.Tryptophan 5-monooxygenase [L-tryptophan, tetrahydropteridine : oxygen oxidoreductase (5-hydroxylating)] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the initial and rate-limiting step in the biosynthesis of the neurotransmitter serotonin [l, 21. Although much effort has been devoted to purification and characterization of tryptophan 5-monooxygenase [3 -1 I], relatively little progress has been made because of the extreme lability of the enzyme with the standard purification procedure. Tong and Kaufman [I 11 reported that they obtained an almost homogenous (85 -90 % pure) preparation of tryptophan 5-monooxygenase from rabbit hindbrain with a 25 yield. However, the specific activity of their final preparation, 2.1 nmol min-' mg-' at 37 "C, was only 58-fold higher than that of the crude enzyme. Recently we purified the rabbit brain tryptophan 5-monooxygenase to a specific activity of 15.9 nmol min-' mg-' at 30 "C with a 29 % yield but it was still far from a homogenous preparation [12], suggesting that much higher purification was required to attain homogenous enzyme.In the present study tryptophan 5-monooxygenase was purified to a specific activity of 374nmol min-' mg-' at 30°C from rat brain-stem and some physical and catalytic properties of the enzyme were examined. This is the first demonstration of the purification of mammalian tryptophan 5-monooxygenase to an apparent homogeneity. EXPERIMENTAL PROCEDURE MaterialsEscherichia coli P-galactosidase, rabbit muscle aldolase rabbit muscle lactate dehydrogenase, beef liver catalase and horse Abbreviations. Me~PteH4, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine; MePteH4, 2-arnino-4-hydroxy-6-methyltetrahydropteridine; Hepes, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid.Enzyme. Tryptophan 5-monooxygenase or tryptophan hydroxylase (EC ...

A calcium-binding protein (protein 10) having a molecular mass of 29 kDa and an isoelectric point of 5.3 was purified from guinea pig brain. The amino acid sequence of fragments from proteolytic digestion of protein 10 revealed an 86% sequence identity with a calcium-binding protein (calretinin) found in chicken retina. Polyclonal antibodies against protein 10 revealed a specific distribution ofthis protein within sensory neurons of auditory, visual, olfactory, nociceptive, and gustatory systems as well as other discrete neuronal circuits in rat and guinea pig brain, whereas no specific label was observed in any of several peripheral tissues examined.