Quantification Of Receptor Expression Research Articles

Cannabinoid 1 (CB1 ) receptor arrestin subtype-selectivity and phosphorylation dependence.

Arrestin or G protein bias may be desirable for novel cannabinoid therapeutics. Arrestin-2 and arrestin-3 translocation to CB1 receptor have been suggested to mediate different functions that may be exploited with biased ligands. Here, the requirement of a recently described phosphorylation motif 'pxxp' (where 'p' denotes phosphorylatable serine or threonine and 'x' denotes any other amino acid) within the CB1 receptor C-terminus for interaction with different arrestin subtypes was examined. Site-directed mutagenesis was conducted to generate nine different phosphorylation-impaired CB1 receptor C-terminal mutants. Bioluminescence resonance energy transfer (BRET) was employed to measure arrestin-2/3 translocation and G protein dissociation of a high efficacy agonist for each mutant. Immunocytochemistry was used to quantify receptor expression. The effects of each mutation were shared for arrestin-2 and arrestin-3 translocation to CB1 receptor pxxp motifs are partially required for arrestin-2/3 translocation, but translocation was not completely inhibited until all phosphorylation sites were mutated. The rate of arrestin translocation was reduced with simultaneous mutation of S425 and S429. Desensitisation of G protein dissociation was inhibited in different mutants proportional to the extent of their respective loss of arrestin translocation. These data do not support the existence of an 'essential' pxxp motif for arrestin translocation to CB1 receptor. These data also identify that arrestin-2 and arrestin-3 have equivalent phosphorylation requirements within the CB1 receptor C-terminus, suggesting arrestin subtype-selective biased ligands may not be viable and that different regions of the C-terminus contribute differently to arrestin translocation.

Angiotensin AT1 and AT2 receptor heteromer expression in the hemilesioned rat model of Parkinson\u2019s disease that increases with levodopa-induced dyskinesia

Background/aimsThe renin-angiotensin system (RAS) is altered in Parkinson’s disease (PD), a disease due to substantia nigra neurodegeneration and whose dopamine-replacement therapy, using the precursor levodopa, leads to dyskinesias as the main side effect. Angiotensin AT1 and AT2 receptors, mainly known for their role in regulating water homeostasis and blood pressure and able to form heterodimers (AT1/2Hets), are present in the central nervous system. We assessed the functionality and expression of AT1/2Hets in Parkinson disease (PD).MethodsImmunocytochemistry was used to analyze the colocalization between angiotensin receptors; bioluminescence resonance energy transfer was used to detect AT1/2Hets. Calcium and cAMP determination, MAPK activation, and label-free assays were performed to characterize signaling in homologous and heterologous systems. Proximity ligation assays were used to quantify receptor expression in mouse primary cultures and in rat striatal sections.ResultsWe confirmed that AT1 and AT2 receptors form AT1/2Hets that are expressed in cells of the central nervous system. AT1/2Hets are novel functional units with particular signaling properties. Importantly, the coactivation of the two receptors in the heteromer reduces the signaling output of angiotensin. Remarkably, AT1/2Hets that are expressed in both striatal neurons and microglia make possible that candesartan, the antagonist of AT1, increases the effect of AT2 receptor agonists. In addition, the level of striatal expression increased in the unilateral 6-OH-dopamine lesioned rat PD model and was markedly higher in parkinsonian-like animals that did not become dyskinetic upon levodopa chronic administration if compared with expression in those that became dyskinetic.ConclusionThe results indicate that boosting the action of neuroprotective AT2 receptors using an AT1 receptor antagonist constitutes a promising therapeutic strategy in PD.

Accounting for pharmacokinetic differences in dual-tracer receptor density imaging

Dual-tracer molecular imaging is a powerful approach to quantify receptor expression in a wide range of tissues by using an untargeted tracer to account for any nonspecific uptake of a molecular-targeted tracer. This approach has previously required the pharmacokinetics of the receptor-targeted and untargeted tracers to be identical, requiring careful selection of an ideal untargeted tracer for any given targeted tracer. In this study, methodology capable of correcting for tracer differences in arterial input functions, as well as binding-independent delivery and retention, is derived and evaluated in a mouse U251 glioma xenograft model using an Affibody tracer targeted to epidermal growth factor receptor (EGFR), a cell membrane receptor overexpressed in many cancers. Simulations demonstrated that blood, and to a lesser extent vascular-permeability, pharmacokinetic differences between targeted and untargeted tracers could be quantified by deconvolving the uptakes of the two tracers in a region of interest devoid of targeted tracer binding, and therefore corrected for, by convolving the uptake of the untargeted tracer in all regions of interest by the product of the deconvolution. Using fluorescently labeled, EGFR-targeted and untargeted Affibodies (known to have different blood clearance rates), the average tumor concentration of EGFR in four mice was estimated using dual-tracer kinetic modeling to be 3.9 ± 2.4 nM compared to an expected concentration of 2.0 ± 0.4 nM. However, with deconvolution correction a more equivalent EGFR concentration of 2.0 ± 0.4 nM was measured.

Advantages of a dual-tracer model over reference tissue models for binding potential measurement in tumors

The quantification of tumor molecular expression in vivo could have a significant impact for informing and monitoring emerging targeted therapies in oncology. Molecular imaging of targeted tracers can be used to quantify receptor expression in the form of a binding potential (BP) if the arterial input curve or a surrogate of it is also measured. However, the assumptions of the most common approaches (reference tissue models) may not be valid for use in tumors. In this study, the validity of reference tissue models is investigated for use in tumors experimentally and in simulations. Three different tumor lines were grown subcutaneously in athymic mice and the mice were injected with a mixture of an epidermal growth factor receptor-targeted fluorescent tracer and an untargeted fluorescent tracer. A one-compartment plasma input model demonstrated that the transport kinetics of both tracers was significantly different between tumors and all potential reference tissues, and using the reference tissue model resulted in a theoretical underestimation in BP of 50% ± 37%. On the other hand, the targeted and untargeted tracers demonstrated similar transport kinetics, allowing a dual-tracer approach to be employed to accurately estimate BP (with a theoretical error of 0.23% ± 9.07%). These findings highlight the potential for using a dual-tracer approach to quantify receptor expression in tumors with abnormal hemodynamics, possibly to inform the choice or progress of molecular cancer therapies.

Non-Invasive Imaging of the Type 2 Cannabinoid Receptor, Focus on Positron Emission Tomography

The type 2 cannabinoid receptor (CB(2)R) is a relatively new target for drug development, as the receptor was only discovered in 1993. The CB(2)R is mainly expressed in tissues and organs related to the immune system. However, in pathological conditions, mostly inflammatory, a strong upregulation has been observed. Because of its expression in activated microglia, the type 2 cannabinoid receptor might play an important role in pathologies with a neuroinflammatory component. Positron emission tomography provides a sensitive non-invasive imaging technique to study and quantify receptor expression. In this review, the importance of CB(2)R imaging, the current status and the future possibilities for the development of CB(2)R PET radioligands are discussed.

Molecular Imaging and Quantitative Measurement of Epidermal Growth Factor Receptor Expression in Live Cancer Cells Using Immunolabeled Gold Nanoparticles

The goal of this study was to assess whether immunolabeled nanoparticle biomarkers are comparable to fluorescent marker imaging in measuring epidermal growth factor receptor (EGFR) expression. EGFR expression was quantified using both imaging methods in four cell lines: A431 human epidermoid carcinoma cells, which are known to have high EGFR expression; two cell lines with lower EGFR expression (270-GBM human glioblastoma xenograft cells and H2224 human glioblastoma xenograft cells); and MDA-MB-453 breast carcinoma cells, which do not express EGFR. To enhance contrast of the nanoparticle biomarkers, a darkfield microspectroscopy system was used that includes a custom epi-illumination light train. Nanoparticle-bound cells were clearly distinguished from control cells not bound to nanoparticles in that they showed a significant increase in detected intensity under darkfield illumination due to nanoparticle scattering. The average nanoparticle-scattering intensity for A431 cells was 41.5 counts per cell compared with 24.7 for 270-GBM cells, 8.77 for H2224 cells, and 0.44 for MDA-MB-453 cells. The average fluorescence intensity for A431 cells was 35.3 counts per cell compared with 28.7 for 270-GBM cells, 5.91 for H2224 cells, and 2.07 for MDA-MB-453 cells. A plot of fluorescence intensity versus nanoparticle-scattering intensity for all four cell lines showed that the data agree with a linear relationship given by the following equation: NP = 1.0691 x FL - 0.3873, where NP is the nanoparticle-scattering intensity and FL is the fluorescence intensity. The covariance of the data with the trend line was R(2) = 0.9409. The average peak wavelength of nanoparticle scattering was 570.93 nm for A431 cells, 565.26 nm for 270-GBM cells, and 562.70 nm for H2224 cells (with no clear peaks observed for MDA-MB-453 cells). This spectral trend shows that nanoparticle scattering may reveal additional information about their nanoenvironment via refractive index sensitivity. Immunolabeled nanoparticles can quantify receptor expression with performance comparable to fluorescence markers and show promise to better characterize receptor expression via their refractive index sensitivity.

Discrepancy in measuring CD4 expression on T‐lymphocytes using fluorescein conjugates in comparison with unimolar CD4‐phycoerythrin conjugates

Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes. However, the results from the use of these different QFC methods vary considerably in the literature. To better identify the causes of these discrepancies, we measured CD4 expression using FITC and phycoerythrin (PE) conjugates to stain CYTO-TROL Control Cells and T-lymphocytes in whole blood and isolated cell preparations. We further examined pH of the cellular microenvironment as a cause of discordant results obtained with the FITC conjugate. Calibration with Quantibrite PE-labeled microspheres and the use of unimolar CD4-PE conjugates provided direct measurement of the antibody bound per cell value (ABC) for CD4 expression on normal T-lymphocytes. Calibration for CD4-FITC monoclonal antibody (Mab) labeled CYTO-TROL Control Cells and normal T-lymphocytes was based on molecules of equivalent soluble fluorochrome (MESF) as determined by FITC-labeled microspheres traceable to NIST RM 8640. The MESF value for CD4-FITC Mab was determined that enabled the conversion of the MESF values obtained for CYTO-TROL cells to ABC. We investigated the likely pH change in the fluorescein microenvironments within FITC-labeled Mab and cells stained with FITC-labeled Mab using a pH sensitive indicator. The mean ABC value for T-lymphocytes prepared from fresh whole blood using CD4-PE conjugate (48,321) was consistent with previous results, and it was much higher than the mean ABC using CD4-FITC Mab (22,156). The mean ABC value for CYTO-TROL cells using CD4-PE conjugate (43,090) was also higher than that using CD4-FITC conjugate (34,734), although the discrepancy was not as great. Further studies suggested the discrepancy in CYTO-TROL results may be accounted for by the low pH of the membrane microenvironment, but the greater discrepancy in T-lymphocytes could not be fully explained. CD4 expression on fresh normal whole blood samples and CYTO-TROL cells can be consistently quantified in ABC units using Quantibrite PE quantification beads and unimolar CD4-PE conjugates. Quantification with CD4-FITC conjugate is not as consistent, but may be improved by the use of CD4 T-cells as biological calibrators. This approximation is valid only for surface receptors with consensus ABC values measured by different QFC methods serving as biological standards.

Prostanoid receptor expression in colorectal cancer related to tumor stage, differentiation and progression

Introduction: Alterations in eicosanoid metabolism is well established in a variety of malignant tumors, particularly colorectal carcinoma. Recent studies in our laboratory have emphasized a role for EP subtype receptors in progression of colorectal cancer and disease specific mortality. Therefore, the aim of the present study was to extend our knowledge to include additional receptor expression (DP1, DP2, FP, IP, TP) for prostanoids (PGD2, TXA2, PGF2α, PGI2) in relationship to tumor stage, differentiation and progression of colorectal cancer. Material and methods: Total RNA from 62 tumors and adjacent normal colon tissue (n = 48) was extracted. Quantification of receptor expression was performed by realtime PCR and related to the expression of an appropriate housekeeping gene (GAPDH). Tumors were assessed according to Dukes A-D (stage I-IV). Results: DP1, DP2, FP and IP receptor subtypes displayed significantly reduced overall expression in tumor tissue compared to normal colon tissue, while the TP receptor subtype showed significantly higher expression in tumor tissue. Overall expression of the prostanoid receptors in tumor tissue was not related to clinical indexes as tumor stage and tumor cell differentiation evaluated by multivariate analyses. Cultured colorectal cancer cell lines with low (HT-29) and high (HCA-7) intrinsic PGE2 production at confluent state did not express DP1 and IP receptor subtypes, but displayed low expression of DP2, FP and TP receptor subtypes. Conclusion: The results in the present study indicate imbalanced expression of prostanoid receptors in colorectal cancer compared to normal colon tissue without clear cut relationship to disease progression. Therefore, future studies should be performed on defined cells within the tumor tissue compartment determining whether any prostanoid receptor(s) is useful as a molecular target in treatment or prevention of colorectal cancer.

Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells

Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes as potential disease biomarkers. CD20 expression in B-cell chronic lymphocytic leukemia (B-CLL) is one of the best examples of such a biomarker, but results from the use of different QFC methods vary considerably. We measured CD20 expression on normal and B-CLL B-cells, using FITC and PE conjugates from the same monoclonal antibody (Mab). As a biological control and calibrator, we also measured CD4 expression on T-cells with FITC and PE Mab. Calibration curves were constructed using the CLSI (formerly NCCLS) consensus guidelines for QFC. Calibration with QuantiBRITE PE-labeled microspheres and the use of unimolar PE conjugates provided direct measurement of antibody bound per cell (ABC) for CD4 and CD20. Calibration for FITC conjugates was based on molecules of equivalent soluble fluorochrome (MESF), as determined by NIST RM 8640 microsphere standards. These MESF values were then converted to ABC, using the CD4 T-cell as a biologic calibrator, to normalize FITC and PE results for CD20 expression. On normal B cells, the mean ABC value for unimolar CD20-PE conjugate was 143,500 (CV +/- 19.1%). The mean ABC value for B-CLL B-cells stained with the same conjugate was 21,700 (CV +/- 42.0%). Using the CD4 T-cell as a biologic calibrator for FITC conjugate, the mean ABC value for CD20-FITC on normal B-cells was 199,300. CD20-FITC staining on B-CLL cells was generally too weak for accurate quantification. On normal T-cells, the mean ABC value for CD4 unimolar PE conjugate was (36,800 +/- 10.4)%, and it did not differ significantly in CLL samples. The expression of CD20 on normal and B-CLL lymphocytes can be quantified in ABC units using unimolar CD20-PE conjugates. In addition, CD4 expression on T-cells can be used as a biological calibrator to quantify CD20-FITC ABC, with reasonable agreement between the two conjugates with different fluorochromes. Issues regarding the accuracy of MESF microsphere calibrators and effective F/P ratios for FITC conjugates will require additional laboratory studies.

Spinal NMDA receptor phosphorylation correlates with the presence of neuropathic signs following peripheral nerve injury in the rat

Substantial evidence has established that activation of the NMDA receptor in the spinal dorsal horn is essential for central sensitization—a phenomenon which comprises various pathophysiological mechanisms underlying neuropathic pain-like signs in animal models. In the present study, a partial sciatic nerve ligation in the rat was used to produce a model of nerve injury-induced pain represented by hypersensitivity to innocuous stimuli (“allodynia”). The aim was to assess whether alteration of NMDA receptor expression correlates with the presence of neuropathic signs. Our approach was to compare spinal NMDA receptor subunit expression and especially subunit 1 phosphorylation, assessed with immunohistochemistry and Western blot at late postoperative times, between nerve-injured rats with marked signs of neuropathy in terms of mechanical and cold hypersensitivity and nerve-injured rats that lacked robust behavioral signs of neuropathy. Quantification of receptor expression was based on comparisons between the dorsal horns ispi- and contralateral to the nerve lesion. The phosphorylated NR1 subunit of the NMDA receptor was found to be significantly increased in the ipsilateral dorsal horn in hypersensitive, but not in non-hypersensitive nerve-injured rats. We did not detect any differences in immunoreactivity in any of the non-phosphorylated NR1, NR2A, NR2B, NR2C or the NR2D subunits. These data suggest that phosphorylation of the NMDA receptor 1 subunit is correlated to the presence of signs of neuropathy (stimulus evoked pain-like behavior) and possibly also to persistent pain following nerve injury. This may represent a mechanism involved in spinal sensitization.

P.1.032 Chronic psychosocial stress in mice decreases the severity of acute dextran sulfate sodium-induced colitis, but impairs regeneration

Substantial evidence has established that activation of the NMDA receptor in the spinal dorsal horn is essential for central sensitization—a phenomenon which comprises various pathophysiological mechanisms underlying neuropathic pain-like signs in animal models. In the present study, a partial sciatic nerve ligation in the rat was used to produce a model of nerve injury-induced pain represented by hypersensitivity to innocuous stimuli (“allodynia”). The aim was to assess whether alteration of NMDA receptor expression correlates with the presence of neuropathic signs. Our approach was to compare spinal NMDA receptor subunit expression and especially subunit 1 phosphorylation, assessed with immunohistochemistry and Western blot at late postoperative times, between nerve-injured rats with marked signs of neuropathy in terms of mechanical and cold hypersensitivity and nerve-injured rats that lacked robust behavioral signs of neuropathy. Quantification of receptor expression was based on comparisons between the dorsal horns ispi- and contralateral to the nerve lesion. The phosphorylated NR1 subunit of the NMDA receptor was found to be significantly increased in the ipsilateral dorsal horn in hypersensitive, but not in non-hypersensitive nerve-injured rats. We did not detect any differences in immunoreactivity in any of the non-phosphorylated NR1, NR2A, NR2B, NR2C or the NR2D subunits. These data suggest that phosphorylation of the NMDA receptor 1 subunit is correlated to the presence of signs of neuropathy (stimulus evoked pain-like behavior) and possibly also to persistent pain following nerve injury. This may represent a mechanism involved in spinal sensitization.

Down-regulation of gamma interferon, tumor necrosis factor type I, interleukin 1 (IL-1) type I, IL-3, IL-4, and transforming growth factor beta type I receptors at the local site during the acute phase of Shigella infection.

An immunohistochemical technique was used to examine whether there was a colocalization of cytokine-specific receptors with cytokine-expressing cells. We have previously shown that there is extensive cytokine production and secretion in the rectal mucosa in shigellosis (interleukin 1 alpha [IL-1 alpha], IL-1 beta, IL-1ra, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha [TNF-alpha], TNF-beta, gamma interferon, granulocyte-macrophage colony-stimulating factor, and transforming growth factor beta [TGF-beta]) (R. Raqib, A. A. Lindberg, B. Wretlind, P. K. Bardhan, U. Andersson, and J. Andersson, Infect. Immun. 63:289-296, 1995; R. Raqib, B. Wretlind, J. Andersson, and A. A. Lindberg, J. Infect. Dis. 171:376-384, 1995). Kinetics for receptor expression was compared with that for cytokine synthesis in the inflamed rectal mucosa from Shigella-infected patients during acute (2 to 6 days after onset of diarrhea) and convalescent (30 to 40 days after onset) stages. Quantification of receptor expression was assessed by computer-assisted analysis of video microscopic images. A selective down-regulation of the receptors for gamma interferon, tumor necrosis factor (TNF receptor [TNFR] type I), IL-1 (IL-1 receptor [IL-1R] types I and type II), IL-3, IL-4, and TGF-beta (TGF-beta receptor type I) was observed at the onset of the disease, with a gradual reappearance during the convalescent stage. However, IL-2R, IL-6R, granulocyte-macrophage colony-stimulating factor receptor, TNFR type II, and TGF-beta receptor type II showed no change in expression during the study period and were comparable to controls. Cytokine receptors were predominantly located to the epithelial layer of the mucosal surface and crypts, with variable expression patterns in the lamina propria. A time-dependent kinetic curve was seen for the soluble IL-2R (sIL-2R), sIL-6R, and sTNFR types I and type II shed in stool at the acute stage similar to that observed for cytokine secretion in stool but at four- to six-times-lower concentration. In contrast, soluble receptor levels in plasma were 100-fold higher than the cytokine levels. The results suggest a dissociation in immune regulation between cytokine production and cytokine receptor expression. The down-regulation of the receptors in acute shigellosis was probably a consequence of cytokine-induced internalization and shedding of the receptors during signal transduction as well as due to programmed regulatory roles played by cytokines and the bacterial antigens.

P-461 - Estimation of KA values by quantification of receptor expression.

P-461 - Estimation of KA values by quantification of receptor expression.

Probing of the expression of the low-density lipoprotein receptor in vivo using an anti-receptor monoclonal antibody

MAC188 is a rat anti-[low-density lipoprotein (LDL) receptor] monoclonal antibody (McAb) which binds to the cell surface receptor with high affinity at physiological temperatures, even in the presence of high concentrations of the natural ligand, LDL. Binding of McAb MAC188 at 37 degrees C is followed by internalization and intracellular sequestering of the receptor, which results in the transient disappearance of the receptor from the cell surface. The high binding affinity and epitope specificity of McAb MAC188 suggested that this antibody could be used to quantify receptor expression in vivo. Mixtures of radiolabelled anti-receptor antibody and a control McAb (MAC221) were injected intravenously into rabbits, and the clearance from serum and uptake into tissues was determined. A fraction of the anti-receptor McAb was cleared rapidly from the circulation by a high-affinity and saturable (receptor-dependent) process. Receptor-dependent uptake of the anti-receptor McAb was measurable in liver, adrenal glands, kidneys, spleen, kidney, thoracic aorta and heart. It was highest in liver and adrenal glands and correlated well with the level of receptor protein and the rate of LDL transport in individual tissues. Anti-receptor McAbs such as MAC188, with suitable domain specificity and binding affinity at physiological temperatures, have important advantages over the natural ligand as tracers for the receptor in vivo, and may find widespread applications in studies of the receptor status (activity) in animals and man.