Despite the presence of the multiple subunits (␣, ␤, ␥, and ␦) and their isoforms for ␥-aminobutyric acid, type A (GABA A ) receptors in mammalian brains, the ␣x␤2␥2 subtypes appear to be the prototype GABA A receptors sharing many properties with native neuronal receptors. In order to gain insight into their subunit stoichiometry and orientation, we prepared a tandem construct of the ␣6 and ␤2 subunit cDNAs where the carboxyl-terminal of ␣6 is linked to the amino-terminal of ␤2 via a linker encoding 10 glutamine residues. Transfection of human embryonic kidney 293 cells with the tandem construct alone failed to induce GABA-dependent Cl ؊ currents, but its cotransfection with the cDNA for ␣6 or ␥2, but not ␤2, led to the appearance of GABA currents which were picrotoxin-sensitive and, in the case of ␥2 containing receptors, responded to a benzodiazepine agonist, U-92330. The high affinity GABA site, however, was detected with [ 3 H]muscimol binding in all combinations of the receptor subunits, including the tandem construct alone or with the ␤2. No appreciable differences were found in their K d (2.5 nM) and B max values (1.4 pmol/mg of protein). These data are consistent with the view that the polypeptides arising from the tandem construct were expressed with the high affinity GABA site, but unable to form GABA channels. The requirement of a specific monomeric subunit (␣6 or ␥2) for the tandem construct to express Cl ؊ currents supports a pentameric structure of GABA A receptors consisting of two ␣6, two ␤2, and one ␥2 for the ␣6␤2␥2 and three ␣6 and two ␤2 for the ␣6␤2 subtype.GABA A 1 receptors, responsible for inhibitory neurotransmission in mammalian brains, are ligand-gated Cl Ϫ channels made of various subunits (␣, ␤, ␥, and ␦) (1-3). Each subunit consists of several isoforms and contains four transmembranespanning segments (M1 to M4) (1-5). Despite the existence of the multiple subunits and their isoforms, combinations of ␣x, ␤2, and ␥2 subunits produced Cl Ϫ channels sharing many functional characteristics with native neuronal receptors and displaying the ability to respond to all the GABA A receptor ligands known up-to-date (1, 3, 6, 7). Such cloned GABA A receptors have been proposed to be of pentameric structure with M2 lining the pore in analogy with another member of the four transmembrane ligand-gated channel family, acetylcholine receptors (1-3). Recent studies, including immunoprecipitation with subunit specific antibodies, have shown the presence of two ␣ subunits per GABA A receptor (8 -11). Further experimental evidence is needed, however, about the stoichiometry of the recombinant GABA A receptors of ␣x␤2␥2 and their modes of association. One way to gain insight into this structural issue is to predetermine the alignment of subunits via gene fusion and to study such fused gene products. Similar approaches have been successful with potassium channels made of their subunits in concatameric or tandem linkages (12,13). In this study we prepared a tandem construct of ␣6 and ␤2 subunit cDNAs of th...

The human 5-hydroxytryptamine-2C (5-HT2C) receptor has been the target of potential anxiolytics and antiobesity drugs, and its positive allosteric modulator was discovered to be l-threo-alpha-d-galacto-octopyranoside, methyl-7-chloro-6,7,8-trideoxy-6-[[(4-undecyl-2-piperidinyl)carbonyl]amino]-1-thiomonohydrochloride (2S-cis) (PNU-69176E). The drug at low micromolar concentrations (<25 microM) markedly enhanced [3H]5-HT binding (more than 300%) by increasing its affinity for low-affinity sites but with no appreciable effect on antagonist ([3H]mesulergine) binding. Functionally, PNU-69176E alone rendered receptors constitutively active, producing the pheno-types of 5-HT-activated receptors, as measured with mesulergine-sensitive guanosine 5'-O-(3-[35S]thio)triphosphate binding, transient inositol 1,4,5-triphosphate release, and [3H]inositol phosphate accumulation. These actions of PNU-69176E were observed with the human 5-HT2C receptor expressed in several mammalian cell lines (human embryonic kidney 293, NIH3T3, and SH-EP) at variable receptor densities (6 to 45 pmol/mg of protein), but not with analogous 5-HT and dopamine receptors (human 5-HT2A, 5-HT2B, 5-HT6, 5-HT7, and dopamine D2-long and D3 receptors). Structurally, PNU-69176E consists of a long alkyl chain and a polar moiety, including the alpha-d-galactopyranoside. Its analogs with shorter alkyl chains (methyl to n-hexyl instead of n-undecyl group) failed to enhance [3H]5-HT binding, and also long alkyl amides are without allosteric modulation. We propose that PNU-69176E may represent a new class of membrane receptor modulators, which probably need a long alkyl chain as a membrane anchor and target a selective polar head group to receptor modulatory sites near the membrane surface.

a human neuroblastoma cell line, SH-SY5Y, produced much greater signals than those expressed in a human embryonic kidney cell line, HEK293. Quinpirole, a prototypic agonist, markedly inhibited forskolin-stimulated cyclic AMP production and Ca 2+ -channel (N-type) currents in SH-SY5Y cells, and enhanced GTPg 35 S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. 3 GTPg 35 S-bound Ga subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Ga-speci®c antibodies. Ga o subunits (not Ga i ) were highly labelled with GTPg 35 S in SH-SY5Y, but not in HEK293 cell membranes, despite their abundance in the both cell types, as shown with reverse transcription-polymerase chain reaction and Western blots. N-type Ca 2+ channels and adenylyl cyclase V (D3-speci®c e ector), on the other hand, exist only in SH-SY5Y cells. 4 More e cient coupling of the D3 receptor to G o subtypes in SH-SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal e ectors only present in SH-SY5Y cells (Ntype Ca 2+ -channels and adenylyl cyclase V). The abundance of G o subtypes in the both cell lines seems to indicate their availability not a limiting factor.

A series of tetrahydropyranyl (THP) derivatives has been developed as potent inhibitors of isoprenylcysteine carboxyl methyltransferase (ICMT) for use as anticancer agents. Structural modification of the submicromolar hit compound 3 led to the potent 3-methoxy substituted analogue 27. Further SAR development around the THP ring resulted in an additional 10-fold increase in potency, exemplified by analogue 75 with an IC(50) of 1.3 nM. Active and potent compounds demonstrated a dose-dependent increase in Ras cytosolic protein. Potent ICMT inhibitors also reduced cell viability in several cancer cell lines with growth inhibition (GI(50)) values ranging from 0.3 to >100 μM. However, none of the cellular effects observed using ICMT inhibitors were as pronounced as those resulting from a farnesyltransferase inhibitor.

No abstract

Human 5-HT7A receptors positively modulated adenylyl cyclases via Gs subtypes of G proteins in human embryonic kidney 293 cells, and bound 5-hydroxytryptamine (HT) with high and low affinity (K(I) values of 1.5 +/- 0.3 and 93 +/- 4 nM). More than 60% of 5-HT7A receptors, however, displayed the high-affinity 5-HT binding with no sensitivity to 5'-guanylylimidodiphosphate. In this study, we found that select amphipathic agents affected the high-affinity 5-HT binding to 5-HT7A. Oleic acid at low concentrations (<15 microM), but not palmitic, stearic, and arachidonic acids, increased maximal [3H]5-HT binding without affecting its K(D) value and [3H]mesulergine (antagonist) binding. Fatty acid-free bovine serum albumin (FF-BSA), a scavenger of fatty acids and lipid metabolites, substantially reduced maximal [3H]5-HT binding (no change in K(D) value and antagonist binding) but lost its action upon treatment with inactive stearic acid. FF-BSA and oleic acid produced no appreciable effects on [3H]5-HT binding to analogous 5-HT receptors 5-HT1D and 5-HT2C. Among various lysophospholipids, lysophosphatidyl choline (50 microM) decreased maximal [3H]5-HT binding, and a similar zwitterion, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; 0.1%), increased it (no change in K(D)). Functionally, 5-HT-induced guanosine-5'-O-(3-[35S]thio)triphosphate (GTPgamma35S) binding was enhanced by oleic acid and CHAPS, but reduced by FF-BSA and lysophosphatidyl choline; the amphipathic agents and FF-BSA did not affect dopamine-induced GTPgamma35S binding at D1, a prototypic Gs-coupled receptor. At 5-HT7A, oleic acid, FF-BSA, CHAPS, and lysophosphatidyl choline also brought about corresponding changes in the half-maximal 5-HT concentration for cAMP production, without affecting the maximal and basal levels. We propose that endogenous, amphipathic lipid metabolites may modulate 5-HT7A receptors allosterically to promote high-affinity 5-HT binding and to enable receptors to couple more efficiently to Gs subtypes of G proteins.

1 Both the 5-HT 1D and 5-HT 1B receptors are implicated in migraine pathophysiology. Recently isochromans have been discovered to bind primate 5-HT 1D receptors with much higher a nity than 5-HT 1B receptors. In the guinea-pig, a primary animal model for anti-migraine drug testing, however, isochromans bound the 5-HT 1D receptor with lower a nity than the gorilla receptor. 2 This species-speci®c pharmacology was investigated, using site-directed mutagenesis on cloned guinea-pig receptors heterologously expressed in human embryonic kidney 293 cells. Mutations of threonine 100 and arginine 102 at the extracellular side of transmembrane II of the guinea-pig 5-HT 1D receptor to the corresponding primate residues, isoleucine and histidine, respectively, enhanced its a nity for isochromans to that of the gorilla receptor, with little e ects on its a nities for serotonin, sumatriptan and metergoline. Free energy change from the R102H mutation was about twice as much as that from the T100I mutation. 3 For G protein-coupling, serotonin marginally enhanced GTPg 35 S binding in membranes expressing the guinea-pig 5-HT 1D receptor and its mutants, but robustly in membranes expressing the gorilla receptor. Sumatriptan enhanced GTPg 35 S binding in the latter nearly as much as serotonin, and several isochromans by 30 ± 60% of serotonin. 4 We discovered key di erences in the function and binding properties of guinea-pig and gorilla 5-HT 1D receptors, and identi®ed contributions of I100 and H102 of primate 5-HT 1D receptors to isochroman binding. Among common experimental animals, only the rabbit shares I100 and H102 with primates, and could be useful for studying isochroman actions in vivo.

1 Cysteine 114 (C114) of the human dopamine D3 receptor is located at the helical face of transmembrane segment III (TMIII) near aspartate 110, a counterion for the amine group of catecholamines. The contributions of C114 to receptor function were investigated here using site-directed mutagenetis of C114 to serine. 2 The C114S mutant, as expressed in Sf-9 cells, bound aminotetralin antagonists (UH-232 and AJ-76) and several agonists ( (7)3-PPP, apomorphine, pramipexole and quinpirole) with markedly lower anities as compared to the wild type D3 receptor, but bound other structurally diverse dopaminergic ligands with only minor changes in anity. Because an N-propyl substituent is the only common structural feature among most aected ligands, we propose that the mutation alters`a propyl cleft' on the receptor. The mutation hardly aected quinpirole-dependent [35 S]-GTPgS binding, suggesting C114 plays a minimal role in receptor-G-protein coupling. 3 N-Ethylmaleimide(NEM), a sulfhydryl modifying agent, blocked ligand binding to the D3 receptor, but not to the C114S mutant. We infer that C114 is the primary residue on the D3 receptor vulnerable to external oxidizing agents. Dopamine D2long and D4 2 receptors contain highly homologous TMIII sequences including an equivalent cysteine residue. However, only the D2long receptor, not the D4 2 receptor, displayed NEM sensitivity similar to that of the D3 receptor. 4 We conclude that C114 is critical for high anity interactions between the D3 receptor and ligands containing an N-propyl substituent, and unlike its counterpart in the D4 2 receptor, is highly susceptible to external oxidizing agents.