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.

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