Allosteric modulation of the human 5-HT(7A) receptor by lipidic amphipathic compounds.

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.

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 GTPgamma(35)S binding and high affinity sites were 3 - 4 fold greater in SH-SY5Y than in HEK293 cells. N-type Ca(2+) 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. GTPgamma(35)S-Bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galpha(i) and Galpha(o) subunits were highly labelled with GTPgamma(35)S in SH-SY5Y cells, but only Galpha(i) subunits were labelled in HEK293 cells. The additional G(o) coupling in SH-SY5Y cells could arise, at least in part, from the presence of G(o) coupled-effectors, such as the N-type Ca(2+) channel, and may contribute to robust agonist-induced GTPgamma(35)S 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.

Cloning of serotonin 5-HT(1) receptor subtypes from the chimpanzee, gorilla and Rhesus monkey and their agonist-induced guanosine 5'gamma(35)S triphosphate binding.

5-HT(1) receptor subtypes ((1B), (1D) and (1F)) have been implicated in migraine pathophysiology and their ligands have been examined for pharmacological actions in various experimental animal models. Considerable divergences exist, however, in their primary sequences between experimental animals and human, and additional models closer to human, such as non-human primates seem to be useful for migraine research. Earlier, we cloned the 5-HT(1D), and here 5-HT(1B) and 5-HT(1F) receptors from the chimpanzee, gorilla and Rhesus monkey, via polymerase chain reactions with their genomic DNAs and primers designed from the corresponding human receptors. Direct sequencing of PCR products showed that the 5-HT(1B) receptors from the chimpanzee, gorilla and monkey differ from the human receptor by 0, 1 and 7 residues, respectively while 5-HT(1F) receptors differ by 0, 3 and 10 residues, respectively. These divergent residues are mostly conservatively substituted and also largely confined to the N-terminal region and the 3rd intracellular loop, away from transmembrane segments and intracellular loops near membrane which are critical for ligand binding and G protein coupling. The chimpanzee 5-HT(1D), 5-HT(1B) and monkey 5-HT(1F) receptors, as heterologously expressed in human embryonic kidney 293 cells, showed robust agonist-induced guanosine 5'gamma (35)S triphosphate (GTPgamma(35)S) binding through activation of G proteins containing Ggamma(i) subunits. Moreover, pronounced inhibition of basal GTPgamma(35)S binding by methiothepin (an antagonist), representing constitutively active receptors, was observed with only 5-HT(1D). Overall, ligand binding and GTPgamma(35)S binding profiles for these primate receptors are comparable to those for the human receptors and validate non-human primates as useful models for human migraine research.

Agonist-induced GTPgamma35S binding mediated by human 5-HT(2C) receptors expressed in human embryonic kidney 293 cells.

The 5-HT(2C) receptor as heterologously expressed in various mammalian cells mediates inositol 1,4,5-triphosphate (IP(3)) signal by activating G(q/11) subtypes of G proteins, but minimally promotes agonist-induced GTPgamma35S binding in membranes due to slow GTP turnover rates of the G proteins. Here we discovered robust (over 200%) agonist-induced GTPgamma35S binding mediated by the human receptor expressed in human embryonic kidney (HEK) 293 cells, and investigated its pharmacology. Agonists concentration-dependently increased GTPgamma35S binding in isolated membranes, which was competitively blocked by antagonists. Intrinsic efficacies of agonists from GTPgamma35S binding were comparable to those from IP(3) measurement. Pertussis toxin treatment largely blocked serotonin-induced GTPgamma35S binding, serotonin high affinity sites by 70% without altering the total binding sites, and reduced IP(3) release by 40%. GTPgamma35S-bound Galpha subunits from serotonin-activated membranes were mainly Galpha(i), judging from immobilization studies with various Galpha-specific antibodies. Inhibition of forskolin-stimulated cAMP formation, however, was not observed. Apparently, the 5-HT(2C) receptor-mediated GTPgamma35S binding is a unique phenotype observed in HEK293 cells, reflecting its coupling to pertussis toxin-sensitive G(i) subtypes, which contribute to the IP(3) signal, along with pertussis toxin-insensitive G(q/11) subtypes.

Efficient functional coupling of the human D3 dopamine receptor to G(o) subtype of G proteins in SH-SY5Y cells.

1 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. 2 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 GTPgamma35S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. 3 GTPgamma35S-bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galphao subunits (not Galphai) were highly labelled with GTPgamma35S 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. 4 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.

Study of cavernosal arterial anatomy using color and power Doppler sonography: impact on hemodynamic parameter measurement.

PURPOSE: We report on color and power Doppler ultrasound to study cavernosal arterial anatomy, and evaluate the impact of vascular anatomy on the measurement of hemodynamic parameters. MATERIALS AND METHODS: Cavernosal arterial anatomy of 42 patients with erectile dysfunction was evaluated using color and power Doppler ultrasound. A computerized waveform analysis was used to measure peak systolic velocity, end diastolic velocity and resistive indexes at various sites, including the penile crura, and proximal mid and distal penile shaft. Hemodynamic parameters were measured in each artery in cases of bifurcated or multiple cavernosal arteries. RESULTS: A total of 80 corpora were adequately evaluated. We observed a single artery without major proximal branches in 37 corpora, a single artery with major proximal branches in 17, bifurcated arteries in 15, 2 cavernosal arteries in 4 and marked arterial tortuosity in 1. In 6 corpora the main cavernosal artery arose from the superficial dorsal artery. The peak systolic velocity was highest at the proximal and decreased progressively at the distal site. The peak systolic velocity plus or minus standard deviation at the mid shaft averaged 69.3+/-30.0% of that at the proximal penile shaft. Of the 15 corpora with bifurcated arteries 67% had a 40% or greater difference in peak systolic velocity between the branches. Complete or partial occlusion of the cavernosal artery was identified in 3 corpora, and a dramatic difference in peak systolic velocity proximal and distal to the stenotic area was demonstrated. CONCLUSIONS: Cavernosal arterial anatomy is variable and hemodynamic parameters differ at various sites of measurement. Parameters should be measured at a consistent proximal site to obtain a reliable assessment. Variations in vascular anatomy and cavernosal artery pathology should be considered when interpreting color Doppler sonography and before penile vascular surgery.

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

1. 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. 2. 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. 3. For G protein-coupling, serotonin marginally enhanced GTPgamma35S binding in membranes expressing the guinea-pig 5-HT1D receptor and its mutants, but robustly in membranes expressing the gorilla receptor. Sumatriptan enhanced GTPgamma35S binding in the latter nearly as much as serotonin, and several isochromans by 30-60% of serotonin. 4. 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.

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 mutagenesis 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]-GTPgammaS 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 affinity 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.

Identification of transmembrane regions critical for ligand binding to the human D3 dopamine receptor using various D3/D1 transmembrane chimeras.

To investigate the roles of individual transmembrane segments (TM) of the human D3 dopamine receptor in its ligand-receptor interactions, we generated chimeric receptors in which its TMs were replaced, one at a time, partially or entirely, by the corresponding TM of the homologous human D1 receptor. Ligand binding properties of the chimeras, as expressed heterologously in Sf9 cells using recombinant baculoviruses, indicate that the critical binding regions for D3-selective (over D1) ligands reside at narrow regions (6 to 8 residues) near the extracellular surface for TMI, II, IV and VI, while TMV seems to be minimally involved in the ligand selectivity. For TMIII and TMVII, the critical regions seem to be deeper, involving at least the 10 residues near the extracellular surface for TMIII, and the entire TM segment for TMVII. This is based on our current observations that the chimeras with the D3 sequence in the critical regions, although the rest of the TM is of D1 origin (except TMVII), showed the binding properties indistinguishable from those of the wild-type receptor. The chimeras with the D1 sequence in the regions, on the other hand, showed ligand binding characteristics wildly variable depending on substituted TMs: Most marked decreases in ligand affinities were observed with the chimeras of TMIII and VII, and intermediate changes with those of TMIV and VI. Replacements of TMV produced no appreciable effects on the affinities of 14 test ligands (except for one). The chimeras of TMI and II with the D1 sequence in the critical regions showed no appreciable specific binding for several radioactive D3-selective ligands, possibly reflecting their critical roles in assembly and folding of the receptor. These critical regions of the D3 receptor were highly homologous to those of the D2 receptor, except for several nonconservatively substituted residues, which could be exploited to develop ligands selective for the D3 over D2 dopamine receptor or vice versa.

Characterization of ligand binding properties of the 5-HT1D receptors cloned from chimpanzee, gorilla and rhesus monkey in comparison with those from the human and guinea pig receptors.

The 5-HT1D receptor is a potential target of anti-migraine drugs, and here its genes were cloned from chimpanzee, gorilla and rhesus monkey, via polymerase chain reactions with their genomic DNAs and the primers designed from the 5' and 3' untranslated regions of the human receptor. Direct sequencing of the polymerase chain reaction (PCR) products revealed high degrees of identity between their deduced amino acid sequences (the chimpanzee, gorilla and rhesus monkey) and that of human, differing by two, four and 11 residues, respectively. The binding properties of the receptors, as expressed in human embryonic kidney 293 cells, were compared to those obtained with the human and guinea pig receptors, the latter differing by 33 residues from the human receptor. Standard serotonergic ligands including several indoles, ergots and methiothepin bound all the cloned primate and guinea pig receptors with comparable, low nanomolar affinities, leading to high correlation coefficients among their Ki values. R(+)-8-Hydroxydipropylaminotetralin, on the other hand, bound the human receptor with the affinity higher than those for the primates and guinea pig receptors. This indicates that certain chemical templates may differentiate the molecular divergences among the 5-HT1D receptors of various animal species, and the use of the non-human primates will be beneficial for pharmacological characterizations, more relevant to the human receptor, of future novel ligands for the 5-HT1D receptor, which are potential anti-migraine drugs.

Agonist-induced [35S]GTPgammaS 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.

Alterations of the benzodiazepine site of rat alpha 6 beta 2 gamma 2-GABAA receptor by replacement of several divergent amino-terminal regions with the alpha 1 counterparts.

1. The benzodiazepine site of the alpha 6 beta 2 gamma 2 subtype of gamma-aminobutyric acidA (GABAA) receptors is distinguishable from that of the alpha 1 beta 2 gamma 2 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 alpha 1 beta 2 gamma 2 subtype. 2. The point mutation of Arg 100 of the alpha 6 subunit to histidine (the corresponding residue in alpha 1) has been shown to enable the alpha 6 beta 2 gamma 2 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 alpha 6 to contain the functional region responsible for the agonistic action of Ro 15-1788. Its replacement with the corresponding alpha 1 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-beta-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 alpha 6, such as R100, V142 and N143, have the ability to influence GABA-dependent Cl- current induction probably by allosterically modulating low affinity GABA sites.

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 (alpha, beta, gamma, and delta) and their isoforms for gamma-aminobutyric acid, type A (GABAA) receptors in mammalian brains, the alpha x beta 2 gamma 2 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 alpha 6 and beta 2 subunit cDNAs where the carboxyl-terminal of alpha 6 is linked to the amino-terminal of beta 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 CI- currents, but its cotransfection with the cDNA for alpha 6 or gamma 2, but not beta 2, led to the appearance of GABA currents which were picrotoxin-sensitive and, in the case of gamma 2 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 beta 2. 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 (alpha 6 or gamma 2) for the tandem construct to express Cl--currents supports a pentameric structure of GABAA receptors consisting of two alpha 6, two beta 2, and one gamma 2 for the alpha 6 beta 2 gamma 2 and three alpha 6 and two beta 2 for the alpha 6 beta 2 subtype.