Biomedical subjects

L Y Yung

Publications and source records attributed to L Y Yung.

18 recordsLinked to original sources

PubMed2001

Chimeric Galphaq subunits can distinguish the long form of the Xenopus Mel1c melatonin receptor from the mammalian mt1 and MT2 melatonin receptors.

The family of melatonin receptors is composed of the mt1, MT2, and Mel1c subtypes. The Mel1c is further divided into one long and two short isoforms. A recent study has shown that, unlike mt1 and MT2, the long form of Mel1c is incapable of activating the pertussis toxin-insensitive G16. Here we used three well-characterized Galphaq chimeras to explore the coupling specificity of the melatonin receptors. The qi5, qo5, and qz5 chimeras can link numerous Gi-coupled receptors to the stimulation of phosphoinositide-specific phospholipase C. Both mt1 and MT2 receptors interacted productively with the Galphaq chimeras, while the long form of Mel1c was totally ineffective. Among the Galphaq chimeras, qo5 was less efficiently coupled to the melatonin receptors. Such differential coupling is best explained by structural differences between the melatonin receptors as well as among the Galphaq chimeras. Since the long form of Mel1c receptor possesses an exceptionally large C-terminal tail, we tested the ability of four melatonin receptor C-terminal tail chimeras (Chi 1-4) to interact with the Galphaq chimeras. The presence of the large C-terminal tail of Mel1c in Chi 1 and Chi 3 markedly hindered their coupling to the Galphaq chimeras. On the other hand, the attachment of either the mtl or MT2 C-terminal tail to a Mel1c backbone produced chimeras (Chi 2 and Chi 4) that were capable of activating the Galphaq chimeras. These findings suggest the involvement of C-terminal regions of melatonin receptors in the recognition of G proteins.

Animals↗

PubMed2001

Galpha(14) links a variety of G(i)- and G(s)-coupled receptors to the stimulation of phospholipase C.

1. The bovine Galpha(14) is a member of the G(q) subfamily of G proteins that can regulate phospholipase Cbeta isoforms but the extent to which Galpha(14) recognizes different receptor classes is not known. 2. Galpha(14) was cotransfected with a variety of receptors in COS-7 cells, and agonist-induced stimulation of phospholipase C was then measured. 3. Activation of the type 2 but not type 1 somatostatin receptor in cells coexpressing Galpha(14) stimulated the accumulation of inositol phosphates; functional expression of both subtypes of somatostatin receptors was determined by the ability of somatostatin to inhibit cyclic AMP accumulation. 4. Among the three opioid receptors (mu, delta, and kappa), only the delta receptor was capable of stimulating IP formation when coexpressed with Galpha(14) in COS-7 cells. 5. A panel of G(i)- and G(s)-linked receptors was screened for their ability to stimulate IP accumulation via Galpha(14). The adenosine A(1), complement C5a, dopamine D(1), D(2) and D(5), formyl peptide, luteinizing hormone, secretin, and the three subtypes of melatonin (mt1, MT2, and Xenopus) receptors were all incapable of activating Galpha(14), while the alpha(2)- and beta(2)-adrenoceptors were able to do so. 6. Galpha(14)-mediated stimulation of phospholipase Cbeta was agonist dose-dependent. These data demonstrate that although Galpha(14) can interact with different classes of receptors, it is much less promiscuous than Galpha(15) or Galpha(16).

Animals↗

PubMed2000

High-molecular-weight kininogen preadsorbed to glass surface markedly reduces neutrophil adhesion.

Adsorbed proteins on biomaterial surfaces determine whether cells adhere, but rheological variables are also critical. Neutrophil adhesion under well-defined radial flow conditions was studied on glass preadsorbed with plasma proteins or plasma protein domain fragments. Fibrinogen, low-molecular-weight kininogen (LK), high-molecular-weight kininogen (HK), cleaved HK (HKa), and recombinant HK domains 3 and 5 (D3 and D5H) were used. The number of adherent cells on the HK and HKa surfaces was less than 10% that found on the fibrinogen absorbed surface. The degree of spreading was minimal and detachment of adherent neutrophils was observed. HK and HKa contain binding sites for both anionic surfaces and neutrophils in the same domain (D5H). When adsorbed to surfaces, HK and HKa did not have the neutrophil binding sites available and therefore exhibited an anti-adhesive effect. Although D5H contains anionic surface binding sites, its small molecular size required a higher number of adsorbed molecules to cover the surface before a significant decrease in cell adhesion was observed. Since LK and D3 do not possess specific anionic surface binding sites, the adsorption of these proteins on glass was very low compared to HK and HKa. Thus, extensive cell adhesion and spreading were observed on the surfaces partially covered with preadsorbed LK and D3.

Adsorption↗

PubMed2000

Regulation of adenylyl cyclase, ERK1/2, and CREB by Gz following acute and chronic activation of the delta-opioid receptor.

Opioid tolerance and physical dependence in mammals can be rapidly induced by chronic exposure to opioid agonists. Recently, opioid receptors have been shown to interact with the pertussis toxin (PTX)-insensitive Gz (a member of the Gi subfamily), which inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell lines expressing delta-opioid receptors with or without Gz to examine the role of Gz in opioid receptor-regulated signaling systems. Each cell line was acutely or chronically treated with [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta-selective agonist, in the absence or presence of PTX. Subsequently, the activities of adenylyl cyclase, cyclic AMP (cAMP)-dependent response element-binding proteins (CREBs), and MAPKs were measured by determining cAMP accumulation and phosphorylation of CREBs and the extracellular signal-regulated protein kinases (ERKs) 1 and 2. In cells coexpressing Gz, DPDPE inhibited forskolin-stimulated cAMP accumulation in a PTX-insensitive manner, but Gz could not replace Gi to mediate adenylyl cyclase supersensitization upon chronic opioid treatment. DPDPE-induced adenylyl cyclase supersensitization was not associated with an increase in the phosphorylation of CREBs. Both Gi and Gz mediated DPDPE-induced activation of ERK1/2, but these responses were abolished by chronic opioid treatment. Collectively, our results show that although Gz mediated opioid-induced inhibition of adenylyl cyclase and activation of ERK1/2, Gz alone was insufficient to mediate opioid-induced adenylyl cyclase supersensitization.

Adenylate Cyclase Toxin↗

PubMed1999

Neutrophil adhesion on polyurethanes preadsorbed with high molecular weight kininogen.

Interaction of biomaterials with blood components including neutrophils is responsible for some of the clinical complications that have occurred in cardiopulmonary bypass, hemodialysis, and ventricular assist procedures. The possibility of inhibiting the initial adhesion of neutrophils to biomaterials has been studied extensively, but the problem remains unsolved. In this study, we investigated the effect of HK adsorption on polyurethane, a widely used component of extracorporeal and intracorporeal devices. HK and HKa were allowed to adsorb on 4 different charged polyurethanes: noncharged (PU), cationic (NR(4)), anionic (SO(3)), and zwitterionic (GPC) polyurethanes. The effect of kininogen adsorption on neutrophil adhesion, the surface density of the adsorbed kininogen, and the exposure of HK domains 3 and 5 (D(3) and D(5H)), which are responsible for the binding of HK to the neutrophil integrin alpha(m)beta(2) or Mac-1, were examined. On PU, NR(4), and SO(3), kininogen adsorption reached 80% of monolayer coverage when 100 pmol/mL or higher concentration of protein solutions were used. The NR(4) surface adsorbed the most kininogen along with a high exposure of D(3) and D(5H). The availability of D(3) and D(5H) allowed neutrophils to bind to the surface via the Mac-1 receptor; thus, on the NR(4) surface, adsorbed kininogens lost their antiadhesive property, which resulted in a high degree of neutrophil adhesion. Increasing Mac-1 expression by exposure to fMLP increased the neutrophil adhesion on this surface. In contrast, exposure of D(3) and D(5H) on SO(3) was significantly less, because HK binds to anionic surfaces with similar protein sequences used for cell binding. This low binding site exposure preserved the antiadhesive property of HK. GPC was resistant to neutrophil adhesion even in the absence of adsorbed kininogens because of its phosphorylcholine moiety. Thus, both SO(3) coupled with kininogen (or kininogen peptides) and GPC have the potential to markedly reduce neutrophil adhesion to biomaterial devices.

Adsorption↗

PubMed1999

The effect of high molecular weight kininogen on neutrophil adhesion to polymer surfaces.

The adhesion of neutrophils on a biomaterial surface depends on the surface chemistry of the material and the cell, as well as the composition and conformation of adsorbed protein and the adherence of other cells when the biomaterial is exposed to circulating blood. In this study, HK and HKa were allowed to adsorb on three different polyurethanes: underivatized (PU-base), quaternized (PU-NR4), sulfonated (PU-SO3). The effect of kininogen adsorption on the degree of neutrophil adhesion was examined. The surface density of the adsorbed protein was also investigated. The PU-NR4 surface adsorbed the most HK and HKa and had the high degree of neutrophil adhesion. Although the surface density of adsorbed HK and HKa on the PU-SO3 surface, the degree of neutrophil on adhesion was significantly lower when compared to the PU-NR4 and PU-base surfaces. HK and HKa contain binding sites for both anionic surfaces and neutrophils in the same domain (D5H). When adsorbed to the anionic PU-SO3 surfaces, HK and HKa did not have the neutrophil binding sites available and therefore, exhibited an anti-adhesive effect. In contrast, the neutrophil binding domains D3 and DsH of adsorbed kininogens were available on the PU-NR4 and PU-base surfaces. Thus, adsorbed kininogens on these two surfaces lost their anti-adhesive property and this led to a high degree of neutrophil adhesion.

Adsorption↗

PubMed1999

Disruption of receptor-mediated activation of G protein by mutating a conserved arginine residue in the switch II region of the alpha subunit.

A naturally occurring point mutation (R231H) within one of the major 3gamma-binding surface (switch II region) on the a subunit of Gs (alpha(s)) has previously been found to disrupt receptor-mediated activation of Gs. The disruption caused by mutating this conserved residue may be a general phenomenon for all a subunits. Homologous mutants of the alpha subunit of Gz [alpha(z); a negative regulator of adenylyl cyclase (AC)] and G16 (alpha16; a stimulator of phospholipase C) were constructed and examined for receptor-mediated regulation of their corresponding effectors. The mutant alphazR209H cannot be fully activated by the delta-opioid receptor, as indicated by the impairment of the inhibition of alpha(s)-stimulated AC and betagamma-mediated stimulation of AC type II (AC2). Similarly, the mutant alpha16R216H lost the ability to mediate receptor-induced activation of phospholipase C and AC2. The receptor coupling efficacy and promiscuity of alpha16R216H were eradicated. The mutation of the conserved arginine has no observable effect on the constitutive activities of the GTPase-deficient derivatives of both alpha(z) and alpha16. The alpha subunit of Gt1 (transducin; alphat1) attenuated betagamma-mediated stimulation of AC2 by sequestrating free betagamma subunits, but the mutant alphat1R204H showed reduced ability to scavenge betagamma-mediated AC2 activation. Presumably, mutation of the conserved arginine disrupted the subunit interactions in addition to the impairment of receptor interaction.

Adenylyl Cyclases↗

PubMed1999

GalphaL1 (Galpha14) couples the opioid receptor-like1 receptor to stimulation of phospholipase C.

In most tissues and cells the opioid receptor-like (ORL1) receptor regulates effectors primarily through the pertussis toxin (PTX)-sensitive guanine nucleotide-binding regulatory proteins (G proteins) Gi/Go. Many Gi-coupled receptors possess additional capability to interact with one or more PTX-insensitive G proteins. Using the betagamma-induced stimulation of type 2 adenylyl cyclase as a readout, we screened the ability of ORL1 receptor to interact with a panel of PTX-insensitive G proteins. In the presence of PTX, activation of the ORL1 receptor resulted in the stimulation of type 2 adenylyl cyclase only in HEK 293 cells coexpressing the alpha subunit of Gz, G12, G14, or G16, but not in cells coexpressing G11, G13, or Gq. Coupling to both Gz and G16 was expected because close relatives of the ORL1 receptor, the opioid receptors, are known to couple productively to these G proteins. ORL1 receptor coupling to either G12 or G14 has not been demonstrated. As predicted by the type 2 adenylyl cyclase assays, activation of the ORL1 receptor resulted in the formation of inositol phosphates in COS-7 cells transiently cotransfected with Galpha14. The ORL1 receptor-mediated stimulation of phospholipase C was found to be Galpha14 dependent, agonist dose dependent, ligand selective, and PTX insensitive. We conclude that G14 can link the ORL1 receptor to regulation of phopholipase C.

Adenylate Cyclase Toxin↗

PubMed1998

The calcitonin gene-related peptide-induced acetylcholinesterase synthesis in cultured chick myotubes is mediated by cyclic AMP.

In vertebrate neuromuscular junctions, post-synaptic specialization includes aggregation of acetylcholine receptors (AChRs) and acetylcholinesterase (AChE). The motor nerve provides soluble factors and electrical activity to achieve this striking localization of AChRs/AChE. Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, is able to stimulate the expression of AChR in cultured myotubes. Similar to AChR regulation, synthesis of AChE in cultured chick myotubes is also stimulated by CGRP. Application of CGRP onto cultured myotubes stimulated the accumulation of intracellular cyclic AMP (cAMP) as well as the expression of AChE mRNA and protein. However, the enzymatic activity of AChE remained unchanged. In cultured myotubes, various drugs affecting the intracellular level of cAMP, such as N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, cholera toxin, and forskolin, could mimic the effect of CGRP in stimulating the expression of AChE. When myotubes were transfected with cDNA encoding constitutively active mutant Galpha(s), the intracellular cAMP synthesis was increased. The increase in cAMP level was in parallel with an increase in the expression of AChE, whereas transfection of active mutant Galpha(i) cDNA decreased the cAMP level as well as the AChE expression. In addition, expression of collagen-tailed AChE was up-regulated by the cAMP pathway. These findings indicated that CGRP-induced AChE regulation is mediated by the cAMP pathway and represented the first evidence to suggest that the regulation of mRNA synthesis of AChR and AChE can be mediated by the same neuron-derived factor.

Acetylcholinesterase↗

PubMed1998

Pertussis toxin-insensitive signaling of the ORL1 receptor: coupling to Gz and G16 proteins.

Nociceptin/OFQ is the endogenous ligand for the G protein-coupled opioid receptor-like (ORL1) receptor. To elucidate the cellular functions of the ORL1 receptor, we examined its ability to interact with Gz and G16, two pertussis toxin (PTX)-insensitive G proteins that are known molecular partners for the opioid receptors. In HEK 293 cells transiently expressing the ORL1 and dopamine D1 receptors, nociceptin/OFQ dose-dependently inhibited dopamine-stimulated cyclic AMP (cAMP) accumulation in a PTX-sensitive manner. However, PTX failed to block the nociceptin/OFQ-induced inhibition of dopamine-stimulated cAMP accumulation in HEK 293 cells co-expressing the alpha-subunit of Gz. This result indicates functional interaction between the ORL1 receptor and Gz. A similar result was obtained with retinoic acid-differentiated SH-SY5Y cells, which endogenously express both the ORL1 receptor and Gz. When the ORL1 receptor was transiently co-expressed in COS-7 cells with the alpha-subunit of G16, nociceptin/OFQ dose-dependently stimulated the formation of inositol phosphates. Nociceptin-induced stimulation of phospholipase C was absolutely dependent on the co-expression of alpha16 and exhibited the appropriate ligand selectivity. In terms of its ability to interact with PTX-insensitive G proteins, the ORL1 receptor behaves very much like the opioid receptors.

Adenylyl Cyclase Inhibitors↗

PubMed1997

Role of amino- and carboxyl-terminal regions of G(alphaZ) in the recognition of Gi-coupled receptors.

Many Gi-coupled receptors are known to interact with the pertussis toxin (PTX)-insensitive Gz protein. Given that the alpha subunits of Gi and Gz share only 60% identity in their amino acid sequences, their receptor-interacting domains must be highly similar. By swapping the carboxyl termini of alpha i2 and alpha z with each other or with those of alpha t, alpha12, and alpha13, we examined the relative contributions of the carboxyl-end 36 amino acids of the alpha chains toward receptor recognition. Chimeric alpha chains lacking the site for PTX-catalyzed ADP-ribosylation were coexpressed with the type II adenylyl cyclase (AC II) and one of several Gi-coupled receptors (formyl peptide, dopamine D2, and delta-opioid receptors) in human embryonic kidney 293 cells. The alpha i2/alpha z chimera was able to interact with both aminergic and peptidergic receptors, resulting in betagamma-mediated stimulation of AC II in the presence of agonists and PTX. Functional and mutational analyses of alpha i2/alpha z revealed that this chimera can inhibit the endogenous ACs of 293 cells. Similarly, the alpha z/alpha i2 chimera seemed to retain the abilities to interact with receptors and inhibit cAMP accumulation. Fusion of the carboxyl-terminal 36 amino acids of alpha z to a backbone of alpha t1 produced a chimera, alpha t1/alpha z, that did not couple to any of the Gi-coupled receptors tested. Interestingly, an alpha13/alpha z chimera (with only the last five amino acids switched) displayed differential abilities to interact with receptors. Signals from aminergic, but not peptidergic, receptors were transduced by alpha13/alpha z. A similar construct, alpha12/alpha z, behaved just like alpha13/alpha z. These results indicated that "alpha i-like" or "alpha z-like" sequences at the carboxyl termini of alpha subunits are not always necessary or sufficient for specifying interaction with Gi-coupled receptors.

Adenylate Cyclase Toxin↗

PubMed1996

Neutrophil adhesion on surfaces preadsorbed with high molecular weight kininogen under well-defined flow conditions.

The adhesion of neutrophils and other leukocytes to biomaterial surfaces is an important phenomenon in the host response to biomaterials because the number of adherent leukocytes is often related to the inflammatory response after implantation. After adhering to biomaterial surfaces, other leukocyte reactions, such as phagocytosis, respiratory burst, and protease release, may occur and result in the deterioration of the implanted biomaterial and injury to peripheral tissue. This study of neutrophil adhesion quantitatively characterizes neutrophil adhesion under well-defined laminar flow conditions using a radial flow chamber. In this rheologically well-defined system, the fluid shear rate on the surface varies continuously with radial position. This allows the study of shear-dependent behavior of neutrophil adhesion. Exploiting the variable shear rate in the radial flow chamber, the kinetics of neutrophil adhesion was obtained using automated video microscopy and image analysis to recursively acquire cell counts from multiple fields in different radial positions, and to quantify the surface density of neutrophil as a function of time. Neutrophil adhesion was studied on glass preadsorbed with fibrinogen and high-molecular-weight kininogen (HK). At a shear rate of 20 s-1, the number of adherent cells on the preadsorbed fibrinogen surface was similar to that on bare glass, and the number of adherent cells on the HK surface was less than 10% of that on the bare glass. We conclude that surfaces preadsorbed with HK are anti-adhesive to neutrophils.

Adsorption↗

PubMed1996

Inverse agonistic effect of ICI-174,864 on the cloned delta-opioid receptor: role of G protein and adenylyl cyclase activation.

Previous studies have established that the delta-selective antagonist ICI-174,864 exhibits negative intrinsic activity at the delta-opioid receptors in NG108-15 membranes. To determine whether ICI-174,864 can function as a true inverse agonist in intact cells, its ability to stimulate cAMP accumulation was examined in a human embryonic kidney 293 cell line (293/DOR) expressing the cloned murine delta-opioid receptor. Forskolin-stimulated cAMP accumulation in the 293/DOR cells was dose-dependently suppressed by the delta-selective agonist [D-Pen2, D-Pen5]enkephalin, and such inhibition was abolished by pertussis toxin or the opiate antagonist naloxone. In contrast, ICI-174,864 significantly potentiated the forskolin response. The ICI-174,864-induced enhancement of the forskolin response exhibited dose-dependency and was antagonized by [D-Pen2,D-Pen5]enkephalin and blocked by pertussis toxin. Neither ICI-174,864 nor pertussis toxin elevated the basal level of cAMP accumulation in the absence of forskolin. Other opiate antagonists, such as naloxone and naltrindole, were ineffective in enhancing the forskolin-stimulated cAMP accumulation. Elevation of cAMP levels in response to the activation of Gs (through either ligand-bound receptor or point mutation on alpha(s)) was also potentiated by ICI-174,864. Our results indicate that ICI-174,864 behaves as an inverse agonist in human embryonic kidney 293 cells stably expressing the delta-opioid receptor. The inverse agonistic effect of ICI-174,864 seemed to require Gi proteins and was clearly manifested when adenylyl cyclase was activated.

Cell Line↗

PubMed1995

Stimulation of cAMP accumulation by the cloned Xenopus melatonin receptor through Gi and Gz proteins.

The Xenopus melatonin receptor was expressed in human embryonic kidney 293 cells and assayed for cAMP accumulation. In transfected 293 cells expressing the melatonin receptor, melatonin dose-dependently inhibited the endogenous adenylyl cyclases. In contrast, melatonin stimulated the accumulation of cAMP in cells co-expressing the type II adenylyl cyclase. Both the inhibitory and stimulatory responses to melatonin were mediated via Gi-like proteins as they were blocked by pertussis toxin. Upon co-transfection with the alpha subunit of Gz, the ability of melatonin to regulate both type II and the endogenous adenylyl cyclases became refractory to pertussis toxin, indicating that the melatonin receptor can also couple to Gz. However, other pertussis toxin-insensitive G proteins such as Gq, G12 and G13 were unable to interact with the melatonin receptor.

Adenylate Cyclase Toxin↗

PubMed1995

Mutant alpha subunit of Gz transforms Swiss 3T3 cells.

Many hormones and neurotransmitters induce cell proliferation by regulating signaling pathways controlled by heterotrimeric G proteins. Mutations that activate the alpha subunits of Gs and Gi2 produce the gsp and gip2 oncogenes that are found in certain human endocrine tumors. Similar mutations have conferred on other G alpha subunits the ability to promote neoplastic transformation in cultured mammalian cells. Gz, a G protein whose normal signaling function is poorly understood, shares with Gi2 the ability to inhibit adenylyl cyclase. We asked whether mutationally activated alpha z can stimulate cell proliferation in a cell line in which stimulation adenylyl cyclase is mitogenic, Swiss 3T3 cells. Stable expression of alpha z-Q205L in Swiss 3T3 cells induced focus formation, a faster growth rate with a higher saturation density, anchorage-independent growth in soft agar, and increased [3H]thymidine incorporation in the absence of growth factors. alpha z-Q205L produced a similar but less extensively transformed phenotype in NIH3T3 cells--increased saturation density in culture, a smaller number of foci and few colonies in soft agar. Stimulation of thymidine incorporation by alpha z-Q205L in Swiss 3T3 cells was increased by co-treatment with cholera toxin, a stimulator of adenylyl cyclase. Taken together, our results indicate that alpha z stimulates one or more mitogenic pathways in Swiss 3T3 cells, and that effectiveness of these mitogenic pathways does not require reducing the concentration of cellular cAMP.

3T3 Cells↗

PubMedDate not supplied

Neutrophil adhesion on phosphorylcholine-containing polyurethanes.

Polyurethanes have been synthesized using glycerophosphorylcholine (GPC) as a chain extender. By altering the ratio of GPC to butanediol (BD), a series of polymers was obtained composed of different contents of phosphorylcholine. Bulk and surface characterization of the polymers was carried out. Differential scanning calorimetry and dynamic mechanical analysis showed that the polymer with the highest phosphorylcholine content (PU-GPC-20) had the lowest soft segment Tg and the highest tensile strength and Young's modulus among the polymers studied. This is due to the high degree of microphase separation in PU-GPC-20 as a result of by ionic aggregation and hydrogen bonding from the zwitterionic phosphorylcholine moiety. PU-GPC-20 contained approximately 20 wt%, of glycerophosphorylcholine. Dynamic contact angle analysis showed that these polymers, especially the ones with high phosphorylcholine content, rearranged themselves to minimize their interfacial tension upon contacting an aqueous environment. Under shear rates of from 20 to 120 s(-1), neutrophils did not adhere to PU-GPC-20. Under similar conditions neutrophil adhesion was observed only at 20 s(-1) on PU-GPC-10, PU-GPC-5 and on the control polyurethane (PU-base). Cell spreading was observed on the control polyurethane but not on any of the other surfaces. The incorporation of phosphorylcholine into the polyurethane backbone effectively reduced neutrophil adhesion and thus potentially could result in lower inflammatory and foreign body responses.

Absorption↗

PubMedDate not supplied

The effect of protein kinase C activation on G(z)-mediated regulation of type 2 and 6 adenylyl cyclases.

Three serine-to-alanine mutants of the alpha subunit of the heterotrimeric G protein G(z) (alpha(z)) were examined for their signaling properties in the presence of phorbol ester treatment. All three alpha(z) mutants resembled wild-type alpha(z) in their abilities to inhibit alpha(s)-stimulated type 6 adenylyl cyclase (AC6) and phorbol ester treatment reduced their magnitudes of inhibition. Depending on the permissive condition, the betagamma-mediated stimulation of type 2 adenylyl cyclase (AC2) was differentially regulated by alpha(z) and the three mutants. Mutation of Ser(27) but not Ser(16) of alpha(z) affected the efficient release of betagamma subunits upon receptor activation and abolished the stimulation of phosphorylated but not alpha(s)-stimulated AC2.

Adenylate Cyclase Toxin↗

PubMedDate not supplied

Immunoglobulin G1 Fc fragment-tagged human opioid receptor-like receptor retains the ability to inhibit cAMP accumulation.

The human opioid receptor-like (ORL(1)) receptor was tagged with the immunoglobulin G1 Fc fragment at the carboxy-terminus and expressed in human embryonic kidney 293 cells. Expression of the ORL(1)-Fc receptor was confirmed by immunohistochemical staining. The fusion protein was enriched by affinity chromatography and then verified by immunodetection. The function of the ORL(1)-Fc receptor was determined by examining nociceptin/OFQ-induced inhibition of cAMP accumulation. The ORL(1)-Fc receptor inhibited the forskolin-stimulated cAMP accumulation. The inhibitory response was selectively induced by nociceptin/OFQ in a dose-dependent and pertussis toxin-sensitive manner. Our results indicate that the carboxy-terminal Fc-tagged ORL(1) receptor retained the ability to interact with G(i) proteins to inhibit adenylyl cyclase.

Adenylate Cyclase Toxin↗