Glen L Alberts

Chloride channel expression with the tandem construct of alpha 6-beta 2 GABAA receptor subunit requires a monomeric subunit of alpha 6 or gamma 2.

Despite the presence of the multiple subunits (α, β, γ, and δ) and their isoforms for γ-aminobutyric acid, type A (GABAA) receptors in mammalian brains, the αxβ2γ2 subtypes appear to be the prototype GABAA 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 α6 and β2 subunit cDNAs where the carboxyl-terminal of α6 is linked to the amino-terminal of β2 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 α6 or γ2, but not β2, led to the appearance of GABA currents which were picrotoxin-sensitive and, in the case of γ2 containing receptors, responded to a benzodiazepine agonist, U-92330. The high affinity GABA site, however, was detected with [3H]muscimol binding in all combinations of the receptor subunits, including the tandem construct alone or with the β2. No appreciable differences were found in their Kd (2.5 nM) and Bmax 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 (α6 or γ2) for the tandem construct to express Cl− currents supports a pentameric structure of GABAA receptors consisting of two α6, two β2, and one γ2 for the α6β2γ2 and three α6 and two β2 for the α6β2 subtype.

Efficient functional coupling of the human D3 dopamine receptor to Go subtype of G proteins in SH-SY5Y cells

The D3 dopamine receptor presumably activates Gi/Go subtypes of G‐proteins, like the structurally analogous D2 receptor, but its signalling targets have not been clearly established due to weak functional signals from cloned receptors as heterologously expressed in mostly non‐neuronal cell lines. In this study, recombinant human D3 receptors expressed in 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 Ca2+‐channel (N‐type) currents in SH‐SY5Y cells, and enhanced GTPγ35S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. GTPγ35S‐bound Gα subunits from quinpirole‐activated and solubilized membranes were monitored upon immobilization with various Gα‐specific antibodies. Gαo subunits (not Gαi) were highly labelled with GTPγ35S 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 Ca2+ channels and adenylyl cyclase V (D3‐specific effector), on the other hand, exist only in SH‐SY5Y cells. More efficient coupling of the D3 receptor to Go subtypes in SH‐SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal effectors only present in SH‐SY5Y cells (N‐type Ca2+‐channels and adenylyl cyclase V). The abundance of Go subtypes in the both cell lines seems to indicate their availability not a limiting factor.

Differential pharmacology between the guinea-pig and the gorilla 5-HT1D receptor as probed with isochromans (5-HT1D-selective ligands).

Both the 5‐HT1D and 5‐HT1B receptors are implicated in migraine pathophysiology. Recently isochromans have been discovered to bind primate 5‐HT1D receptors with much higher affinity than 5‐HT1B receptors. In the guinea‐pig, a primary animal model for anti‐migraine drug testing, however, isochromans bound the 5‐HT1D receptor with lower affinity than the gorilla receptor. This species‐specific 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‐HT1D receptor to the corresponding primate residues, isoleucine and histidine, respectively, enhanced its affinity for isochromans to that of the gorilla receptor, with little effects on its affinities for serotonin, sumatriptan and metergoline. Free energy change from the R102H mutation was about twice as much as that from the T100I mutation. For G protein‐coupling, serotonin marginally enhanced GTPγ35S binding in membranes expressing the guinea‐pig 5‐HT1D receptor and its mutants, but robustly in membranes expressing the gorilla receptor. Sumatriptan enhanced GTPγ35S binding in the latter nearly as much as serotonin, and several isochromans by 30–60% of serotonin. We discovered key differences in the function and binding properties of guinea‐pig and gorilla 5‐HT1D receptors, and identified contributions of I100 and H102 of primate 5‐HT1D 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.

Agonist-induced GTPγ35S binding mediated by human 5-HT2C receptors expressed in human embryonic kidney 293 cells

Abstract The 5-HT2C receptor as heterologously expressed in various mammalian cells mediates inositol 1,4,5-triphosphate (IP3) signal by activating Gq/11 subtypes of G proteins, but minimally promotes agonist-induced GTPγ35S binding in membranes due to slow GTP turnover rates of the G proteins. Here we discovered robust (over 200%) agonist-induced GTPγ35S binding mediated by the human receptor expressed in human embryonic kidney (HEK) 293 cells, and investigated its pharmacology. Agonists concentration-dependently increased GTPγ35S binding in isolated membranes, which was competitively blocked by antagonists. Intrinsic efficacies of agonists from GTPγ35S binding were comparable to those from IP3 measurement. Pertussis toxin treatment largely blocked serotonin-induced GTPγ35S binding, serotonin high affinity sites by 70% without altering the total binding sites, and reduced IP3 release by 40%. GTPγ35S-bound Gα subunits from serotonin-activated membranes were mainly Gαi, judging from immobilization studies with various Gα-specific antibodies. Inhibition of forskolin-stimulated cAMP formation, however, was not observed. Apparently, the 5-HT2C receptor-mediated GTPγ35S binding is a unique phenotype observed in HEK293 cells, reflecting its coupling to pertussis toxin-sensitive Gi subtypes, which contribute to the IP3 signal, along with pertussis toxin-insensitive Gq/11 subtypes.

Agonist-induced [35S]GTPγS binding in the membranes of Spodoptera frugiperda insect cells expressing the human D3 dopamine receptor

In the membranes of Spodoptera frugiperda (Sf-9) insect cells heterologously expressing the human D3 dopamine receptor, agonists selective for the receptor, but not antagonists, robustly enhanced [35S]GTPgammaS binding. Quinpirole, for instance, dose-dependently enhanced [35S]GTPgammaS binding with a half-maximal concentration of 2.3 +/- 0.2 nM. Its action was absent in the cells infected with wild type viruses, and competitively blocked by an antagonist, YM-09151-2. A number of known agonists enhanced [35S]GTPgammaS binding to variable degrees, probably reflecting their differential efficacy to activate target G-proteins via the receptor. This agonist-induced [35S]GTPgammaS binding was abolished by N-ethylmaleimide, a selective blocking agent for Gi/Go proteins, with no appreciable effect on ligand binding. We propose coupling of the cloned D3 receptor to endogenous G-proteins in Sf-9 cells, probably homologs of mammalian Gi/Go proteins. Despite the apparent coupling of the D3 receptor to G-proteins, GTPgammaS (10 microM) failed to decrease agonist binding ([3H]dopamine) to the D3 receptor, probably due to small affinity differences between low and high affinity states for agonists in the D3 receptor, as well as due to high receptor density in Sf-9 cells. We conclude that agonist-induced [35S]GTPgammaS binding for the D3 receptor is suitable for estimating ligand intrinsic efficacy and pharmacological characterizations of ligand-receptor interactions.

Contributions of cysteine 114 of the human D3 dopamine receptor to ligand binding and sensitivity to external oxidizing agents

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 ((−)3‐PPP, apomorphine, pramipexole and quinpirole) with markedly lower affinities as compared to the wild type D3 receptor, but bound other structurally diverse dopaminergic ligands with only minor changes in affinity. Because an N‐propyl substituent is the only common structural feature among most affected ligands, we propose that the mutation alters ‘a propyl cleft’ on the receptor. The mutation hardly affected quinpirole‐dependent [35S]‐GTPγS 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 D42 receptors contain highly homologous TMIII sequences including an equivalent cysteine residue. However, only the D2long receptor, not the D42 receptor, displayed NEM sensitivity similar to that of the D3 receptor. 4 We conclude that C114 is critical for high affinity interactions between the D3 receptor and ligands containing an N‐propyl substituent, and unlike its counterpart in the D42 receptor, is highly susceptible to external oxidizing agents.

Advantages of heterologous expression of human D2long dopamine receptors in human neuroblastoma SH-SY5Y over human embryonic kidney 293 cells

The human D2long dopamine receptor when expressed heterologously in a human neuronal cell line, SH‐SY5Y, produced more robust functional signals than when expressed in a human embryonic kidney cell line, HEK293. Quinpirole (agonist)‐induced GTPγ35S binding and high affinity sites were 3–4 fold greater in SH‐SY5Y than in HEK293 cells. N‐type Ca2+ channel currents present in SH‐SY5Y cells, but not HEK293 cells, were inhibited potently by quinpirole with a half‐maximal inhibitory concentration of 0.15±0.03 nM. Inhibition of adenylyl cyclases by agonists, on the other hand, was of similar potency and efficacy in the two cell lines. GTPγ35S‐Bound Gα subunits from quinpirole‐activated and solubilized membranes were monitored upon immobilization with various Gα‐specific antibodies. Gαi and Gαo subunits were highly labelled with GTPγ35S in SH‐SY5Y cells, but only Gαi subunits were labelled in HEK293 cells. The additional Go coupling in SH‐SY5Y cells could arise, at least in part, from the presence of Go coupled‐effectors, such as the N‐type Ca2+ channel, and may contribute to robust agonist‐induced GTPγ35S binding, which is a reliable means for measuring ligand intrinsic efficacy. It appears that expression of neuronal G protein‐coupled receptors in neuronal environments could reveal additional functional characteristics that are absent in non‐neuronal cell lines. This appears to be due to, at least in part, to the presence of neuron‐specific effectors. These findings underscore the importance of the cellular environment in which drug actions are examined, particularly in the face of intensive efforts to develop drugs for G protein‐coupled receptors of various origins.

Alterations of the benzodiazepine site of rat α6β2γ2-GABAA receptor by replacement of several divergent amino-terminal regions with the αl counterparts

1 The benzodiazepine site of the α6β2γ2 subtype of γ‐aminobutyric acidA (GABAA) receptors is distinguishable from that of the α1β2γ2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15–1788, which behaves as an antagonist in the α1β2γ2 subtype. 2 The point mutation of Arg 100 of the α6 subunit to histidine (the corresponding residue in α1) has been shown to enable the α6β2γ2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15–1788 to enhance GABA‐induced Cl− currents. 3 Here we identified the segment of P161 to L187 of α6 to contain the functional region responsible for the agonistic action of Ro 15–1788. Its replacement with the corresponding α1 sequence abolished the ability of Ro 15–1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U‐92330 and 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others. 4 In addition we have learned that several residues in the N‐terminal region of α6, such as R100, V142 and N143, have the ability to influence GABA‐dependent Cl current induction probably by allosterically modulating low affinity GABA sites.

A unique phenotype of 5‐HT2C, agonist‐induced GTPγ35S binding, transferable to 5‐HT2A and 5‐HT2B, upon swapping intracellular regions

The human 5‐HT2C receptor, when expressed heterologously in various mammalian cell lines (HEK293, SH‐EP and NIH‐3T3) at various receptor densities (6 to 45 pmol mg−1 protein), mediates robust agonist‐induced GTPγ35S binding from coupling to Gi subtypes of G proteins, in addition to Gq/11. Such a phenotype, however, was not seen with the human 5‐HT2A and 5‐HT2B receptors, indicating their common pathway with 5‐HT2C limited to Gq/11, not including Gi. Because intracellular regions are largely responsible for signalling pathways, we prepared the chimeras of the 5‐HT2A and 5‐HT2B receptors where the second and third intracellular loops, and the C‐terminal region were replaced with the 5‐HT2C counterparts. The chimeras showed robust agonist‐induced GTPγ35S binding. Relative intrinsic efficacies of agonists from the GTPγ35S binding were nearly identical to the reported values for their parent receptors as measured with Ca2+ or [3H]‐inositol phosphate accumulation. Also the chimeras displayed the same ligand‐binding properties as the parent receptors. We conclude that the phenotype of agonist‐induced GTPγ35S binding is unique to 5‐HT2C among the 5‐HT2 receptor family, and is transferable to 5‐HT2A and 5‐HT2B, upon swapping intracellular sequences, without altering their receptor pharmacology.

Discovery of a protein sequence in the N-terminal region of the human neuronal α7 nicotinic acetylcholine receptor involved in homomeric interactions.

The neuronal nicotinic acetylcholine receptor subunit, alpha7, can form homopentameric receptor/ion channel complexes. Potential contributions of its N-terminal region to homomeric interactions were investigated, in comparison with the corresponding region of an analogous heteromeric subunit, alpha3. Recombinant chimeras were prepared upon engineering the N-terminal alpha7 (M1-V224) or alpha3 (M1-S232) sequence into the background of another homomeric mouse 5-hydroxytryptamine3 (5-HT)(3) receptor. The alpha7/5-HT(3) chimera, when expressed heterologously in a human epithelial cell line, SH-EP1, robustly expressed alpha-bungarotoxin binding sites as homooligomers while the alpha3/5-HT(3) did not produce epibatidine (non-selective ligand) binding sites, and did not interfere the alpha7/5-HT3 phenotype, upon co-expression. Yeast two hybrid assays with the N-terminal regions showed positive responses between alpha7:alpha7, but not between alpha7:alpha3 and alpha3:alpha3. Similar assays with the alpha7 N-terminal region and its five smaller fragments (G23-N46, D47-N90, V91-N133, S134-M182and Q183-V224) revealed that the G23-N46 sequence is involved in homomeric interactions. Replacement of the corresponding region of the alpha3/5-HT(3) chimera with the alpha7 G23-N46 sequence conferred a dominant negative role on the chimera, by abolishing the alpha7/5-HT(3) phenotype. These results support the view that the G23-N46 portion of the alpha7 N-terminal region may contribute to receptor homooligomerizations.