TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP

Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (M_f). The apparent diffusion coefficient (D_app) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a “membrane fence” model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more “fluid” and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of G_q/G₁₁ family.In conclusion: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R–G_q/G₁₁α protein coupling.     

Disruption of the Plasma Membrane Integrity by Cholesterol Depletion Impairs Effectiveness of TRH Receptor-Mediated Signal Transduction via Gq/G11α Proteins

We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G_q/11α proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G₁₁α. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G_q/11α was assessed by agonist-stimulated [³⁵S]GTPγS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca²⁺ response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G_q/11α proteins.     

Visualization of agonist-induced association and trafficking of green fluorescent protein-tagged forms of both beta-arrestin-1 and the thyrotropin-releasing hormone receptor-1.

A fusion protein (beta-arrestin-1-green fluorescent protein (GFP)) was constructed between beta-arrestin-1 and a modified form of the green fluorescent protein from Aequorea victoria. Expression in HEK293 cells allowed immunological detection of an 82-kDa cytosolic polypeptide with antisera to both beta-arrestin-1 and GFP. Transient expression of this construct in HEK293 cells stably transfected to express the rat thyrotropin-releasing hormone receptor-1 (TRHR-1) followed by confocal microscopy allowed its visualization evenly distributed throughout the cytoplasm. Addition of thyrotropin-releasing hormone (TRH) caused a profound and rapid redistribution of beta-arrestin-1-GFP to the plasma membrane followed by internalization of beta-arrestin-1-GFP into distinct, punctate, intracellular vesicles. TRH did not alter the cellular distribution of GFP transiently transfected into these cells nor the distribution of beta-arrestin-1-GFP following expression in HEK293 cells lacking the receptor. To detect potential co-localization of the receptor and beta-arrestin-1 in response to agonist treatment, beta-arrestin-1-GFP was expressed stably in HEK293 cells. A vesicular stomatitis virus (VSV)-tagged TRHR-1 was then introduced transiently. Initially, the two proteins were fully resolved. Short term exposure to TRH resulted in their plasma membrane co-localization, and sustained exposure to TRH resulted in their co-localization in punctate, intracellular vesicles. In contrast, beta-arrestin-1-GFP did not relocate or adopt a punctate appearance in cells that did not express VSV-TRHR-1. Reciprocal experiments were performed, with equivalent results, following transient expression of beta-arrestin-1 into cells stably expressing VSVTRHR-1-GFP. These results demonstrate the capacity of beta-arrestin-1-GFP to interact with the rat TRHR-1 and directly visualizes their recruitment from cytoplasm and plasma membrane respectively into overlapping, intracellular vesicles in an agonist-dependent manner.     

Subcellular shifts of trimeric G-proteins following activation of Baker’s yeast by glucose

Addition of glucose to a resting cell suspension of the yeastSaccharomyces cerevisiae was accompanied by marked shifts of the Gα-protein subunits from the plasma membrane to the cell interior. This process was rapid with half-times between <10 and 20 s. The decrease of the plasma membrane pool of the G_iα/G_oα- and G_qα/G₁₁α-protein subunits correlated with an increase in acid-sensitive forms of these proteins which was recovered in the mitochondrial and/or lysosomal membrane fraction. In contrast to cells from higher organisms glucose-stimulated yeast exhibits an extremely rapid type of the redistribution (internalization). The question remains’open as to the functional significance of the internalized forms of the G-proteins as these remain sequestered from the plasma membrane. well after glucose has been consumed.     

Chromatography studies on bioaffinity of nine ligands of α1 adrenoceptor to α1D subtypes overexpressed in cell membrane

To improve selectivity and specificity of cell membrane chromatography (CMC), the chromatography affinities of nine ligands of α₁-adrenergic receptor(AR)to α_1D-AR subtype were investigated. The human embryonic kidney (HEK) 293 cells expressed by cDNA of α_1D-AR subtypes were cultured and cell membrane stationary phase (CMSP) was prepared. Then the interactions between ligands and α_1D-AR in CMSP were investigated using CMC. The affinity rank order to α_1D-AR subtype obtained from CMC for the nine α₁-adrenoceceptor ligands is: prazosin, BMY7378, phentolamine, oxymetazoline, 5-methylurapidil, norepinephrine, phenylephrine, methoxamine, RS-17053. The affinity rank order is similar and correlates well with that obtained from others’ radioligand binding assays (RBA). CMSP prepared by transfected HEK293 cells with α_1D-adrenoceptor cDNA and CMC method could be used to evaluate affinities of drug-receptor and drug-receptor subtypes and to screen drugs selective to α_1D-AR.     

Activation of cloned BK<Subscript>Ca</Subscript> channels in nitric oxide-induced apoptosis of HEK293 cells

The large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK_Ca has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK_Ca channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the α-subunit of BK_Ca channel, hSloα, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloα cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloα cells exhibited functional features similar to native BK_Ca channel in VSMCs. Exposuring of HEK- hSloα cells to S-nitroso-N-acetyl-penicillamine increased the hSloα channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloα channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 μM NS1619, the specific agonist of BK_Ca, independently increased hSloα currents and induced apoptosis. These results indicated that activation of cloned BK_Ca channel was involved in NO-induced apoptosis of HEK293 cells.     

Molecular cloning, stable expression and cellular localization of human α1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of α1D -adrenergic receptor

The cDNAs encoding for three subtypes of adrenergic receptors, α_1A-, α_1B- and α_1D-ARs, were cloned and expressed in HEK 293 cells. Expression of α_1A- and α_1B-AR subtypes in HEK 293 cells was stable even with increased passages but that of α_1D-AR was not. Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of α_1A- and α_1B-ARs was primarily localized on the cell membrane whereas expression of α_1D-AR was␣predominantly intracellular. Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing α_1D-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of α_1D-AR to the cell membrane and thus, significantly improving its stability and availability for ligand binding studies.Sunil M. Khattar, Roop Singh Bora and Priyanka Priyadarsiny contributed equally to this work.     

Suppression of adenylyl cyclase-mediated cAMP production by plasma membrane associated cytoskeletal protein 4.1G

It has been shown lately that activity of G protein-coupled receptors (GPCRs) is regulated by an array of proteins binding to carboxy (C)-terminus of GPCRs. Proteins of 4.1 family are subsets of subcortical cytoskeletal proteins and are known to stabilize cellular structures and proteins at the plasma membrane. One of the 4.1 family proteins, 4.1G has been shown to interact with the C-terminus of GPCRs and regulate intracellular distribution of the receptors, including parathyroid hormone (PTH)/PTH-related protein receptor (PTHR). PTHR is coupled to trimeric G proteins G_s and G_q, which activate the adenylyl cyclase/cyclic AMP (cAMP) pathway and phospholipase C pathway, respectively. During the course of investigation of the role of 4.1G on adenylyl cyclase/cAMP signaling pathway, we found that 4.1G suppressed forskolin-induced cAMP production in cells. The cAMP accumulation induced by forskolin was decreased in HEK293 cells overexpressing 4.1G or increased in 4.1G-knockdown cells. Furthermore, PTH -(1-34)-stimulated cAMP production was also suppressed in the presence of exogenously expressed 4.1G despite its activity to increase the distribution of PTHR to the cell surface. In cells overexpressing FERM domain-deleted 4.1G, a mutant form of the protein deficient in plasma membrane distribution, neither forskolin-induced nor PTH -(1-34)-stimulated cAMP production was not altered. The suppression of the forskolin-induced cAMP production was observed even in membrane preparations of 4.1G-overexpressing cells. In 4.1G-knockdown HEK293 cells, plasma membrane distribution of adenylyl cyclase 6, one of the major subtypes of the enzyme in the cells, showed a slight decrease, in spite of the increased production of cAMP in those cells when stimulated by forskolin. Also, cytochalasin D treatment did not cause any influence on forskolin-induced cAMP production in HEK293 cells. These data indicate that plasma membrane-associated 4.1G regulates GPCR-mediated G_s signaling by suppressing adenylyl cyclase-mediated cAMP production.     

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated,Low-Density Plasma Membrane Fragments

Principal Findings

HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C³⁵¹I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025–0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [³²P]GTPase and [³⁵S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound G_i1α within δ-opioid receptor-G_i1α fusion protein, but it was also valid for PTX-sensitive G proteins of G_i/G_o family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the “wobble in cone” model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye.

Summary

Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.     

TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins

Objectives: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to G_q/11 proteins.

Methods: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP).

Results: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of G_q/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of G_iα1, β-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of G_iα1 and β-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling.

Conclusion: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.     

Resolution of G(s)alpha and G(q)alpha/G(11)alpha proteins in membrane domains by two-dimensional electrophoresis: the effect of long-term agonist stimulation

Low-density membrane-domain fractions were prepared from S49 lymphoma cells and clone e2m11 of HEK293 cells expressing a large number of thyrotropin-releasing hormone receptor (TRH-R) and G(11)alpha by flotation on sucrose density gradients. The intact cell structure was broken by detergent-extraction, alkaline-treatment or drastic homogenization. Three types of low-density membranes were resolved by two-dimensional electrophoresis and analyzed for G(s)alpha (S49) or G(q)alpha/G11) (e2m11) content. Four individual immunoblot signals of Gsalpha protein were identified in S49 lymphoma cells indicating complete resolution of the long G(s)alpha L+/-ser and short G(s)alpha S+/-ser variants of G(s)alpha. All these were diminished by prolonged agonist (isoprenaline) stimulation. In e2m11-HEK cells, five different immunoblot signals were detected indicating post-translational modification of G proteins of G(q)alpha/G(11)alpha family. The two major spots corresponding to exogenously (over)expressed G(11)alpha and endogenous G(q)alpha were reduced; the minor spots diminished by hormonal stimulation. Parallel analysis by silver staining of the total protein content indicated that no major changes in protein composition occurred under these conditions. Our data thus indicate that agonist-stimulation of target cells results in down-regulation of all different members of G(s) and G(q)/G(11) families. This agonist-specific effect may be demonstrated in crude membrane as well as domain/raft preparations and it is not accompanied by changes in overall protein composition.     

G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E₂), couple to the G proteins G_s and G_i/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E₂ and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of G_i/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of G_i/o and retains receptors in the plasma membrane.     

Agonist-induced tyrosine phosphorylation of Gq/G11 alpha requires the intact structure of membrane domains

Stimulation of receptors coupled to G(q)/G(11) protein may induce phosphorylation on a tyrosine residue of the alpha subunit of this G protein, which is an essential event for G(q)/G(11) activation. Here we observed that in HEK293 cells stably expressing high levels of thyrotropin-releasing hormone (TRH) receptors and G(11)alpha protein the maximal tyrosine phosphorylation of G(q)/G(11)alpha was reached within 10 min of TRH stimulation and then it faded away at longer time periods of agonist exposure. The G(q)/G(11)alpha protein levels did not change during this treatment. Incubation of intact cells with beta-cyclodextrin (beta CD) for 40 min prior to hormone exposure significantly decreased the rapid transient tyrosine phosphorylation. Subsequent replenishment of cholesterol levels reversed the former negative effect of beta CD. Isolation of caveolin-enriched, detergent-resistant membrane domains indicated destruction of these structures in beta CD-treated cells. These data indicate that the preserved integrity of plasma membrane domains/caveolae is required for complete agonist-induced phosphorylation of G(q)/G(11)alpha.     

Disruption of the plasma membrane integrity by cholesterol depletion impairs effectiveness of TRH receptor-mediated signal transduction via G(q)/G(11)alpha proteins

We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G(q/11)alpha proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G(11)alpha. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G(q/11)alpha was assessed by agonist-stimulated [(35)S]GTPgammaS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca(2+) response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G(q/11)alpha proteins.     

Stimulation and conformational change of G<Subscript>o</Subscript>α induced by GAP-43

GAP-43 and G_o are peripheral membrane proteins enriched in neuronal growth cone. GAP-43 was highly purified from bovine cerebral cortex and myristoylated G_oα was highly purified from Escherichia coli cotransformed with pQE60 G_oα and pBB131 (NMT). GAP-43 stimulated GTPγS binding to G_oα and the stimulation effect was dependent on concentration of GAP-43. Protein-protein binding experiments using CaM-Sepharose affinity media revealed that G_oα GDP bound GAP-43 directly to form intermolecular complex. This interaction induced conformational change of G_oα. In the presence of GAP-43, fluorescence spectrum of G_oα GDP blue shifted 4 nm; fluorescence intensity increased 35.3% and apparent quenching constant (Ksv) increased from (1.1 ±0.22) ×10⁵ to (4.1±0.43) × 10⁵ (M⁻¹). However, no obvious changes of fluorescence spectra of G_oα GTPγS were observed in the absence or presence of GAP-43. Our results indicated that GAP-43 induced conformational change of G_oα GDP so as to accelerate GDP release and subsequent GTPγS binding, which activates G proteins to trigger signal transduction and amplification. These results provided insights into understanding the function of G proteins in coupling between receptors and effectors and the key role of GDP/GTP exchange mode in GTPase cycle.     

Identification and functional characterization of natural human melanocortin 1 receptor mutant alleles in Pakistani population

Melanocortin 1 receptor (MC1R), a Gs protein‐coupled receptor of the melanocyte's plasma membrane, is a major determinant of skin pigmentation and phototype. Upon activation by α‐melanocyte stimulating hormone, MC1R triggers the cAMP cascade to stimulate eumelanogenesis. We used whole‐exome sequencing to identify causative alleles in Pakistani families with skin and hair hypopigmentation. Six MC1R mutations segregated with the phenotype in seven families, including a p.Val174del in‐frame deletion and a p.Tyr298* nonsense mutation, that were analyzed for function in heterologous HEK293 cells. p.Tyr298* MC1R showed no agonist‐induced signaling to the cAMP or ERK pathways, nor detectable agonist binding. Conversely, signaling was comparable for p.Val174del and wild‐type in HEK cells overexpressing the proteins, but binding analysis suggested impaired cell surface expression. Flow cytometry and confocal imaging studies revealed reduced plasma membrane expression of p.Val174del and p.Tyr298*. Therefore, p.Tyr298* was a total loss‐of‐function (LOF) allele, while p.Val174del displayed a partial LOF attribute.     

Multiple G proteins compete for binding with the human gonadotropin releasing hormone receptor

The GnRH receptor is coupled to G proteins of the families G_q and G₁₁. G_q and G₁₁ coupling leads to intracellular signaling through the phospholipase C pathway. GnRHR coupling to other G proteins is controversial. This study provides evidence that G protein families G_s, G_i, G_q and G₁₁ complete for binding with the GnRHR. We quantified interactions of over-expressed G proteins with GnRHR by a competitive binding approach, using measurements of second messengers, IP and cAMP. Transient co-transfection of HEK293 cells with human WT GnRHR and with stimulatory and inhibitory G proteins (G_q, G₁₁ and G_s, G_i) led to either production or inhibition of total inositol phosphate (IP) production, depending on the G protein that was over-expressed. Studies were conducted in different human (COS7, HeLa) and rodent-derived (CHO-K1, GH₃) cell lines in order to confirm that G protein promiscuity observed with the GnRHR was not limited to a particular cell type.     

Dominant portion of thyrotropin-releasing hormone receptor is excluded from lipid domains. Detergent-resistant and detergent-sensitive pools of TRH receptor and Gqalpha/G11alpha protein

Some G protein-coupled receptors might be spacially targetted to discrete domains within the plasma membrane. Here we assessed the localization in membrane domains of the epitope-tagged, fluorescent version of thyrotropin-releasing hormone receptor (VSV-TRH-R-GFP) expressed in HEK293 cells. Our comparison of three different methods of cell fractionation (detergent extraction, alkaline treatment/sonication and mechanical homogenization) indicated that the dominant portion of plasma membrane pool of the receptor was totally solubilized by Triton X-100 and its distribution was similar to that of transmembrane plasma membrane proteins (glycosylated and non-glycosylated forms of CD147, MHCI, CD29, CD44, transmembrane form of CD58, Tapa1 and Na,K-ATPase). As expected, caveolin and GPI-bound proteins CD55, CD59 and GPI-bound form of CD58 were preferentially localized in detergent-resistant membrane domains (DRMs). Trimeric G proteins G(q)alpha/G(11)alpha, G(i)alpha1/G(i)alpha2, G(s)alphaL/G(s)alphaS and Gbeta were distributed almost equally between detergent-resistant and detergent-solubilized pools. In contrast, VSV-TRH-R-GFP, Galpha, Gbeta and caveolin were localized massively only in low-density membrane fragments of plasma membranes, which were generated by alkaline treatment/sonication or by mechanical homogenization of cells. These data indicate that VSV-TRH-R-GFP as well as other transmembrane markers of plasma membranes are excluded from TX-100-resistant, caveolin-enriched membrane domains. Trimeric G protein G(q)alpha/G(11)alpha occurs in both DRMs and in the bulk of plasma membranes, which is totally solubilized by TX-100.     

Production and characterization of an antiserum which recognizes the native receptor for thyrotropin-releasing hormone.

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa and a minor broad band at 100-120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy.     

A2A R mediated modulation in IP3 levels altering the [Ca2+]i through cAMP-dependent PKA signalling pathway

Stimulation of A_2A receptors (A_2A R) coupled to G_s/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of A_2A R in the modulation of free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) involving IP₃, cAMP and PKA was investigated in HEK 293-A_2A R. The levels of IP₃ and cAMP were observed by enzyme immunoassay detection method and [Ca²⁺]_i using Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with A_2A R agonist NECA showed increased levels of cAMP, PKA, IP₃ and [Ca²⁺]_i levels. However, the reverse effect was observed with A_2A R antagonists (ZM241385 and caffeine). Blocking the G_αq/PLC/DAG/IP₃ pathway with neomycin, a PLC inhibitor did not affect the modulation of IP₃ and [Ca²⁺]_i levels in HEK 293-A_2A R cells. To investigate the G_αi/AC/cAMP/PKA, HEK 293-A_2A R cells pre-treated with pertussis toxin followed by forskolin in the presence of A_2A R agonist (NECA) showed no effect on cAMP levels. Further, G_αs/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IP₃ mediated [Ca²⁺]_i modulation. In the HEK 293-A_2A R cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IP₃ and [Ca²⁺]_i levels. The study distinctly demonstrated that A_2A R modulates IP₃ levels to release the [Ca²⁺]_i via cAMP-dependent PKA. The role of A_2A R mediated G_αs pathway inducing IP₃ mediated [Ca²⁺]_i release may open new avenues in the therapy of neurodegenerative disorder.