TY  - JOUR
AU  - Mavri, Maša
AU  - Glišić, Sanja
AU  - Senćanski, Milan
AU  - Vrecl, Milka
AU  - Rosenkilde, Mette M.
AU  - Spiess, Katja
AU  - Kubale, Valentina
PY  - 2023
UR  - https://vinar.vin.bg.ac.rs/handle/123456789/10682
AB  - The viral G-protein-coupled receptor (vGPCR) BILF1 encoded by the Epstein–Barr virus (EBV) is an oncogene and immunoevasin and can downregulate MHC-I molecules at the surface of infected cells. MHC-I downregulation, which presumably occurs through co-internalization with EBV-BILF1, is preserved among BILF1 receptors, including the three BILF1 orthologs encoded by porcine lymphotropic herpesviruses (PLHV BILFs). This study aimed to understand the detailed mechanisms of BILF1 receptor constitutive internalization, to explore the translational potential of PLHV BILFs compared with EBV-BILF1.
T2  - Cellular and Molecular Biology Letters
T1  - Patterns of human and porcine gammaherpesvirus-encoded BILF1 receptor endocytosis
VL  - 28
IS  - 1
SP  - 14
DO  - 10.1186/s11658-023-00427-y
ER  - 

@article{
author = "Mavri, Maša and Glišić, Sanja and Senćanski, Milan and Vrecl, Milka and Rosenkilde, Mette M. and Spiess, Katja and Kubale, Valentina",
year = "2023",
abstract = "The viral G-protein-coupled receptor (vGPCR) BILF1 encoded by the Epstein–Barr virus (EBV) is an oncogene and immunoevasin and can downregulate MHC-I molecules at the surface of infected cells. MHC-I downregulation, which presumably occurs through co-internalization with EBV-BILF1, is preserved among BILF1 receptors, including the three BILF1 orthologs encoded by porcine lymphotropic herpesviruses (PLHV BILFs). This study aimed to understand the detailed mechanisms of BILF1 receptor constitutive internalization, to explore the translational potential of PLHV BILFs compared with EBV-BILF1.",
journal = "Cellular and Molecular Biology Letters",
title = "Patterns of human and porcine gammaherpesvirus-encoded BILF1 receptor endocytosis",
volume = "28",
number = "1",
pages = "14",
doi = "10.1186/s11658-023-00427-y"
}

Mavri, M., Glišić, S., Senćanski, M., Vrecl, M., Rosenkilde, M. M., Spiess, K.,& Kubale, V.. (2023). Patterns of human and porcine gammaherpesvirus-encoded BILF1 receptor endocytosis. in Cellular and Molecular Biology Letters, 28(1), 14.
https://doi.org/10.1186/s11658-023-00427-y

Mavri M, Glišić S, Senćanski M, Vrecl M, Rosenkilde MM, Spiess K, Kubale V. Patterns of human and porcine gammaherpesvirus-encoded BILF1 receptor endocytosis. in Cellular and Molecular Biology Letters. 2023;28(1):14.
doi:10.1186/s11658-023-00427-y .

Mavri, Maša, Glišić, Sanja, Senćanski, Milan, Vrecl, Milka, Rosenkilde, Mette M., Spiess, Katja, Kubale, Valentina, "Patterns of human and porcine gammaherpesvirus-encoded BILF1 receptor endocytosis" in Cellular and Molecular Biology Letters, 28, no. 1 (2023):14,
https://doi.org/10.1186/s11658-023-00427-y . .

TY  - JOUR
AU  - Susec, Maja
AU  - Senćanski, Milan V.
AU  - Glišić, Sanja
AU  - Veljković, Nevena V.
AU  - Pedersen, Christina
AU  - Drinovec, Luka
AU  - Stojan, Jurij
AU  - Nøhr, Jane
AU  - Vrecl, Milka
PY  - 2019
UR  - https://vinar.vin.bg.ac.rs/handle/123456789/8490
AB  - This study aimed to functionally characterize β2-adrenergic (β2AR) and insulin receptor (IR) heteromers in regard to β-arrestin 2 (βarr2) recruitment and cAMP signaling and to examine the involvement of the cytoplasmic portion of the IR β chain in heteromerization with β2AR. Evidence for β2AR:IR:βarr2 complex formation and the specificity of the IR:βarr2 interaction was first provided by bioinfomatics analysis. Receptor-heteromer investigation technology (HIT) then provided functional evidence of β2AR:IR heterodimerization by showing isoproterenol-induced but not insulin-induced GFP2-βarr2 recruitment to the β2AR:IR complex; the IR:βarr2 interaction was found to only be constitutive. The constitutive IR:βarr2 BRET signal (BRETconst) was significantly smaller in cells coexpressing IR-RLuc8 and a GFP2-βarr2 1–185 mutant lacking the proposed IR binding domain. β2AR:IR heteromerization also influenced the pharmacological phenotype of β2AR, i.e., its efficacy in recruiting βarr2 and activating cAMP signaling. Evidence suggesting involvement of the cytoplasmic portion of the IR β chain in the interaction with β2AR was provided by BRET2 saturation and HIT assays using an IR 1–1271 stop mutant lacking the IR C-terminal tail region. For the complex consisting of IR 1–1271–RLuc8:β2AR-GFP2, saturation was not reached, most likely reflecting random collisions between IR 1–1271 and β2AR. Furthermore, in the HIT assay, no substantial agonist-induced increase in the BRET2 signal was detected that would be indicative of βarr2 recruitment to the IR 1–1271:β2AR heteromer. Complementary 3D visualization of β2AR:IR provided supporting evidence for stability of the heterotetramer complex and identified amino acid residues involved in β2AR:IR heteromerization. © 2019
T2  - Neuropharmacology
T1  - Functional characterization of β2-adrenergic and insulin receptor heteromers
VL  - 152
SP  - 78
EP  - 89
DO  - 10.1016/j.neuropharm.2019.01.025
ER  - 

@article{
author = "Susec, Maja and Senćanski, Milan V. and Glišić, Sanja and Veljković, Nevena V. and Pedersen, Christina and Drinovec, Luka and Stojan, Jurij and Nøhr, Jane and Vrecl, Milka",
year = "2019",
abstract = "This study aimed to functionally characterize β2-adrenergic (β2AR) and insulin receptor (IR) heteromers in regard to β-arrestin 2 (βarr2) recruitment and cAMP signaling and to examine the involvement of the cytoplasmic portion of the IR β chain in heteromerization with β2AR. Evidence for β2AR:IR:βarr2 complex formation and the specificity of the IR:βarr2 interaction was first provided by bioinfomatics analysis. Receptor-heteromer investigation technology (HIT) then provided functional evidence of β2AR:IR heterodimerization by showing isoproterenol-induced but not insulin-induced GFP2-βarr2 recruitment to the β2AR:IR complex; the IR:βarr2 interaction was found to only be constitutive. The constitutive IR:βarr2 BRET signal (BRETconst) was significantly smaller in cells coexpressing IR-RLuc8 and a GFP2-βarr2 1–185 mutant lacking the proposed IR binding domain. β2AR:IR heteromerization also influenced the pharmacological phenotype of β2AR, i.e., its efficacy in recruiting βarr2 and activating cAMP signaling. Evidence suggesting involvement of the cytoplasmic portion of the IR β chain in the interaction with β2AR was provided by BRET2 saturation and HIT assays using an IR 1–1271 stop mutant lacking the IR C-terminal tail region. For the complex consisting of IR 1–1271–RLuc8:β2AR-GFP2, saturation was not reached, most likely reflecting random collisions between IR 1–1271 and β2AR. Furthermore, in the HIT assay, no substantial agonist-induced increase in the BRET2 signal was detected that would be indicative of βarr2 recruitment to the IR 1–1271:β2AR heteromer. Complementary 3D visualization of β2AR:IR provided supporting evidence for stability of the heterotetramer complex and identified amino acid residues involved in β2AR:IR heteromerization. © 2019",
journal = "Neuropharmacology",
title = "Functional characterization of β2-adrenergic and insulin receptor heteromers",
volume = "152",
pages = "78-89",
doi = "10.1016/j.neuropharm.2019.01.025"
}

Susec, M., Senćanski, M. V., Glišić, S., Veljković, N. V., Pedersen, C., Drinovec, L., Stojan, J., Nøhr, J.,& Vrecl, M.. (2019). Functional characterization of β2-adrenergic and insulin receptor heteromers. in Neuropharmacology, 152, 78-89.
https://doi.org/10.1016/j.neuropharm.2019.01.025

Susec M, Senćanski MV, Glišić S, Veljković NV, Pedersen C, Drinovec L, Stojan J, Nøhr J, Vrecl M. Functional characterization of β2-adrenergic and insulin receptor heteromers. in Neuropharmacology. 2019;152:78-89.
doi:10.1016/j.neuropharm.2019.01.025 .

Susec, Maja, Senćanski, Milan V., Glišić, Sanja, Veljković, Nevena V., Pedersen, Christina, Drinovec, Luka, Stojan, Jurij, Nøhr, Jane, Vrecl, Milka, "Functional characterization of β2-adrenergic and insulin receptor heteromers" in Neuropharmacology, 152 (2019):78-89,
https://doi.org/10.1016/j.neuropharm.2019.01.025 . .

TY  - JOUR
AU  - Senćanski, Milan V.
AU  - Glišić, Sanja
AU  - Šnajder, Marko
AU  - Veljković, Nevena V.
AU  - Poklar Ulrih, Nataša
AU  - Mavri, Janez
AU  - Vrecl, Milka
PY  - 2019
UR  - https://vinar.vin.bg.ac.rs/handle/123456789/8648
AB  - This study aimed to design and functionally characterize peptide mimetics of the nanobody (Nb) related to the β2-adrenergic receptor (β2-AR) (nanobody-derived peptide, NDP). We postulated that the computationally derived and optimized complementarity-determining region 3 (CDR3) of Nb is sufficient for its interaction with receptor. Sequence-related Nb-families preferring the agonist-bound active conformation of β2-AR were analysed using the informational spectrum method (ISM) and β2-AR:NDP complexes studied using protein-peptide docking and molecular dynamics (MD) simulations in conjunction with metadynamics calculations of free energy binding. The selected NDP of Nb71, designated P3, was 17 amino acids long and included CDR3. Metadynamics calculations yielded a binding free energy for the β2-AR:P3 complex of ΔG = (−7.23 ± 0.04) kcal/mol, or a Kd of (7.9 ± 0.5) μM, for T = 310 K. In vitro circular dichroism (CD) spectropolarimetry and microscale thermophoresis (MST) data provided additional evidence for P3 interaction with agonist-activated β2-AR, which displayed ~10-fold higher affinity for P3 than the unstimulated receptor (MST-derived EC50 of 3.57 µM vs. 58.22 µM), while its ability to inhibit the agonist-induced interaction of β2-AR with β-arrestin 2 was less evident. In summary, theoretical and experimental evidence indicated that P3 preferentially binds agonist-activated β2-AR. © 2019, The Author(s).
T2  - Scientific Reports
T1  - Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR)
VL  - 9
IS  - 1
SP  - 16555
DO  - 10.1038/s41598-019-52934-8
ER  - 

@article{
author = "Senćanski, Milan V. and Glišić, Sanja and Šnajder, Marko and Veljković, Nevena V. and Poklar Ulrih, Nataša and Mavri, Janez and Vrecl, Milka",
year = "2019",
abstract = "This study aimed to design and functionally characterize peptide mimetics of the nanobody (Nb) related to the β2-adrenergic receptor (β2-AR) (nanobody-derived peptide, NDP). We postulated that the computationally derived and optimized complementarity-determining region 3 (CDR3) of Nb is sufficient for its interaction with receptor. Sequence-related Nb-families preferring the agonist-bound active conformation of β2-AR were analysed using the informational spectrum method (ISM) and β2-AR:NDP complexes studied using protein-peptide docking and molecular dynamics (MD) simulations in conjunction with metadynamics calculations of free energy binding. The selected NDP of Nb71, designated P3, was 17 amino acids long and included CDR3. Metadynamics calculations yielded a binding free energy for the β2-AR:P3 complex of ΔG = (−7.23 ± 0.04) kcal/mol, or a Kd of (7.9 ± 0.5) μM, for T = 310 K. In vitro circular dichroism (CD) spectropolarimetry and microscale thermophoresis (MST) data provided additional evidence for P3 interaction with agonist-activated β2-AR, which displayed ~10-fold higher affinity for P3 than the unstimulated receptor (MST-derived EC50 of 3.57 µM vs. 58.22 µM), while its ability to inhibit the agonist-induced interaction of β2-AR with β-arrestin 2 was less evident. In summary, theoretical and experimental evidence indicated that P3 preferentially binds agonist-activated β2-AR. © 2019, The Author(s).",
journal = "Scientific Reports",
title = "Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR)",
volume = "9",
number = "1",
pages = "16555",
doi = "10.1038/s41598-019-52934-8"
}

Senćanski, M. V., Glišić, S., Šnajder, M., Veljković, N. V., Poklar Ulrih, N., Mavri, J.,& Vrecl, M.. (2019). Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR). in Scientific Reports, 9(1), 16555.
https://doi.org/10.1038/s41598-019-52934-8

Senćanski MV, Glišić S, Šnajder M, Veljković NV, Poklar Ulrih N, Mavri J, Vrecl M. Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR). in Scientific Reports. 2019;9(1):16555.
doi:10.1038/s41598-019-52934-8 .

Senćanski, Milan V., Glišić, Sanja, Šnajder, Marko, Veljković, Nevena V., Poklar Ulrih, Nataša, Mavri, Janez, Vrecl, Milka, "Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR)" in Scientific Reports, 9, no. 1 (2019):16555,
https://doi.org/10.1038/s41598-019-52934-8 . .

TY  - CONF
AU  - Senćanski, Milan
AU  - Vrecl, Milka
AU  - Veljković, Nevena V.
AU  - Glišić, Sanja
PY  - 2018
UR  - https://vinar.vin.bg.ac.rs/handle/123456789/11013
AB  - Stabilization of specific G-protein coupled receptor (GPCR) conformation is achieved by ligand binding to orthosteric or allosteric sites on a GPCRs. A crucial unresolved issue in GPCRs activation/signaling is the role of receptor structural conformations in G protein/effector protein selection. One of the possible approaches to get comprehensive depiction of GPCRs activation dynamics are molecular simulations and recently described nanobody-derived intrabodies. Monomeric single-domain antibody (nanobody) from the Camelid family was found to allosterically bind to and stabilizes distinct conformational states of the β2AR. By applying informational spectrum method (ISM), a virtual spectroscopy method for investigation of the protein-protein interactions, we have designed peptide mimetic of the nanobody related to the β2AR (nanobody derived peptide, NDP). Further, interaction between NDP and the ligand-bound β2AR active conformation have been studied by protein-peptide docking, molecular dynamics simulations and metadynamics calculations of free energy binding. Finally, the affinity of selected NDPs towards agonist-activated β2AR was also studied by microscale thermophoresis (MST) and by bioluminescence resonance energy transfer (BRET) based β-arrestin 2 recruitment assay. MST data predicted micromolar range interaction of selected NDPs with the β2AR, while the preliminary β-arrestin 2 recruitment results suggest prospective further modification and optimization of NDPs toward effective modulators of the β2AR.
PB  - Department of Biology and Ecology : Faculty of Sciences University of Novi Sad
C3  - Biologia Serbica : Belgrade BioInformatics Conference : BelBi2018 : program and the book of abstracts; June 18-22
T1  - Combined in silico and experimental approach to identify the peptide mimetic of the nanobody that stabilize functional conformational state of the beta2 adrenergic receptor (β2AR)
VL  - 40
IS  - 1
SP  - 58
UR  - https://hdl.handle.net/21.15107/rcub_vinar_11013
ER  - 

@conference{
author = "Senćanski, Milan and Vrecl, Milka and Veljković, Nevena V. and Glišić, Sanja",
year = "2018",
abstract = "Stabilization of specific G-protein coupled receptor (GPCR) conformation is achieved by ligand binding to orthosteric or allosteric sites on a GPCRs. A crucial unresolved issue in GPCRs activation/signaling is the role of receptor structural conformations in G protein/effector protein selection. One of the possible approaches to get comprehensive depiction of GPCRs activation dynamics are molecular simulations and recently described nanobody-derived intrabodies. Monomeric single-domain antibody (nanobody) from the Camelid family was found to allosterically bind to and stabilizes distinct conformational states of the β2AR. By applying informational spectrum method (ISM), a virtual spectroscopy method for investigation of the protein-protein interactions, we have designed peptide mimetic of the nanobody related to the β2AR (nanobody derived peptide, NDP). Further, interaction between NDP and the ligand-bound β2AR active conformation have been studied by protein-peptide docking, molecular dynamics simulations and metadynamics calculations of free energy binding. Finally, the affinity of selected NDPs towards agonist-activated β2AR was also studied by microscale thermophoresis (MST) and by bioluminescence resonance energy transfer (BRET) based β-arrestin 2 recruitment assay. MST data predicted micromolar range interaction of selected NDPs with the β2AR, while the preliminary β-arrestin 2 recruitment results suggest prospective further modification and optimization of NDPs toward effective modulators of the β2AR.",
publisher = "Department of Biology and Ecology : Faculty of Sciences University of Novi Sad",
journal = "Biologia Serbica : Belgrade BioInformatics Conference : BelBi2018 : program and the book of abstracts; June 18-22",
title = "Combined in silico and experimental approach to identify the peptide mimetic of the nanobody that stabilize functional conformational state of the beta2 adrenergic receptor (β2AR)",
volume = "40",
number = "1",
pages = "58",
url = "https://hdl.handle.net/21.15107/rcub_vinar_11013"
}

Senćanski, M., Vrecl, M., Veljković, N. V.,& Glišić, S.. (2018). Combined in silico and experimental approach to identify the peptide mimetic of the nanobody that stabilize functional conformational state of the beta2 adrenergic receptor (β2AR). in Biologia Serbica : Belgrade BioInformatics Conference : BelBi2018 : program and the book of abstracts; June 18-22
Department of Biology and Ecology : Faculty of Sciences University of Novi Sad., 40(1), 58.
https://hdl.handle.net/21.15107/rcub_vinar_11013

Senćanski M, Vrecl M, Veljković NV, Glišić S. Combined in silico and experimental approach to identify the peptide mimetic of the nanobody that stabilize functional conformational state of the beta2 adrenergic receptor (β2AR). in Biologia Serbica : Belgrade BioInformatics Conference : BelBi2018 : program and the book of abstracts; June 18-22. 2018;40(1):58.
https://hdl.handle.net/21.15107/rcub_vinar_11013 .

Senćanski, Milan, Vrecl, Milka, Veljković, Nevena V., Glišić, Sanja, "Combined in silico and experimental approach to identify the peptide mimetic of the nanobody that stabilize functional conformational state of the beta2 adrenergic receptor (β2AR)" in Biologia Serbica : Belgrade BioInformatics Conference : BelBi2018 : program and the book of abstracts; June 18-22, 40, no. 1 (2018):58,
https://hdl.handle.net/21.15107/rcub_vinar_11013 .

TY  - JOUR
AU  - Kopanja, Lazar
AU  - Kovačević, Zorana
AU  - Tadić, Marin
AU  - Žužek, Monika Cecilija
AU  - Vrecl, Milka
AU  - Frangež, Robert
PY  - 2018
UR  - http://link.springer.com/10.1007/s00418-018-1670-0
UR  - https://vinar.vin.bg.ac.rs/handle/123456789/7762
AB  - Detailed shape analysis of cells is important to better understand the physiological mechanisms of toxins and determine their effects on cell morphology. This study aimed to develop a procedure for accurate morphological analysis of cell shape and use it as a tool to estimate toxin activity. With the aim of optimizing the method of cell morphology analysis, we determined the influence of ostreolysin A and pleurotolysin B complex (OlyA/PlyB) on the morphology of murine neuronal NG108-15 cells. A computational method was introduced and successfully applied to quantify morphological attributes of the NG108-15 cell line before and after 30 and 60 min exposure to OlyA/PlyB using confocal microscopy. The modified circularity measure (Formula presented.) for shape analysis was applied, which defines the degree to which the shape of the neuron differs from a perfect circle. It enables better detection of small changes in the shape of cells, making the outcome easily detectable numerically. Additionally, we analyzed the influence of OlyA/PlyB on the cell area, allowing us to detect the cells with blebs. This is important because the formation of plasma membrane protrusions such as blebs often reflects cell injury that leads to necrotic cell death. In summary, we offer a novel analytical method of neuronal cell shape analysis and its correlation with the toxic effects of the pore-forming OlyA/PlyB toxin in situ.
T2  - Histochemistry and Cell Biology
T1  - Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex
VL  - 150
IS  - 1
SP  - 93
EP  - 102
DO  - 10.1007/s00418-018-1670-0
ER  - 

@article{
author = "Kopanja, Lazar and Kovačević, Zorana and Tadić, Marin and Žužek, Monika Cecilija and Vrecl, Milka and Frangež, Robert",
year = "2018",
abstract = "Detailed shape analysis of cells is important to better understand the physiological mechanisms of toxins and determine their effects on cell morphology. This study aimed to develop a procedure for accurate morphological analysis of cell shape and use it as a tool to estimate toxin activity. With the aim of optimizing the method of cell morphology analysis, we determined the influence of ostreolysin A and pleurotolysin B complex (OlyA/PlyB) on the morphology of murine neuronal NG108-15 cells. A computational method was introduced and successfully applied to quantify morphological attributes of the NG108-15 cell line before and after 30 and 60 min exposure to OlyA/PlyB using confocal microscopy. The modified circularity measure (Formula presented.) for shape analysis was applied, which defines the degree to which the shape of the neuron differs from a perfect circle. It enables better detection of small changes in the shape of cells, making the outcome easily detectable numerically. Additionally, we analyzed the influence of OlyA/PlyB on the cell area, allowing us to detect the cells with blebs. This is important because the formation of plasma membrane protrusions such as blebs often reflects cell injury that leads to necrotic cell death. In summary, we offer a novel analytical method of neuronal cell shape analysis and its correlation with the toxic effects of the pore-forming OlyA/PlyB toxin in situ.",
journal = "Histochemistry and Cell Biology",
title = "Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex",
volume = "150",
number = "1",
pages = "93-102",
doi = "10.1007/s00418-018-1670-0"
}

Kopanja, L., Kovačević, Z., Tadić, M., Žužek, M. C., Vrecl, M.,& Frangež, R.. (2018). Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex. in Histochemistry and Cell Biology, 150(1), 93-102.
https://doi.org/10.1007/s00418-018-1670-0

Kopanja L, Kovačević Z, Tadić M, Žužek MC, Vrecl M, Frangež R. Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex. in Histochemistry and Cell Biology. 2018;150(1):93-102.
doi:10.1007/s00418-018-1670-0 .

Kopanja, Lazar, Kovačević, Zorana, Tadić, Marin, Žužek, Monika Cecilija, Vrecl, Milka, Frangež, Robert, "Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex" in Histochemistry and Cell Biology, 150, no. 1 (2018):93-102,
https://doi.org/10.1007/s00418-018-1670-0 . .

TY  - JOUR
AU  - Mandić, Maja
AU  - Drinovec, Luka
AU  - Glišić, Sanja
AU  - Veljković, Nevena V.
AU  - Nohr, Jane
AU  - Vrecl, Milka
PY  - 2014
UR  - https://vinar.vin.bg.ac.rs/handle/123456789/216
AB  - Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and beta(2)-adrenergic receptor (beta(2)AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on beta(2)AR properties, we showed that IR coexpression had no effect on the ligand binding properties of beta(2)AR; however, IR reduced beta(2)AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between beta(2)AR and IR, we employed quantitative BRET 2 saturation and competition assays. Saturation assay data suggested constitutive beta(2)AR and IR homo-and heteromerization. Calculated acceptor/donor (AD(50)) values as a measure of the relative affinity for homo-and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET2 signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET2 data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of beta(2)AR and IR identified intracellular domains encompassing residues at the end of the 7th TM domain and C-terminal tail of beta(2)AR and a cytoplasmic part of the IR beta chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order beta(2)AR: IR oligomer formation, likely comprising heteromers of homodimers.
T2  - PLOS One
T1  - Demonstration of a Direct Interaction between beta(2)-Adrenergic Receptor and Insulin Receptor by BRET and Bioinformatics
VL  - 9
IS  - 11
DO  - 10.1371/journal.pone.0112664
ER  - 

@article{
author = "Mandić, Maja and Drinovec, Luka and Glišić, Sanja and Veljković, Nevena V. and Nohr, Jane and Vrecl, Milka",
year = "2014",
abstract = "Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and beta(2)-adrenergic receptor (beta(2)AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on beta(2)AR properties, we showed that IR coexpression had no effect on the ligand binding properties of beta(2)AR; however, IR reduced beta(2)AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between beta(2)AR and IR, we employed quantitative BRET 2 saturation and competition assays. Saturation assay data suggested constitutive beta(2)AR and IR homo-and heteromerization. Calculated acceptor/donor (AD(50)) values as a measure of the relative affinity for homo-and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET2 signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET2 data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of beta(2)AR and IR identified intracellular domains encompassing residues at the end of the 7th TM domain and C-terminal tail of beta(2)AR and a cytoplasmic part of the IR beta chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order beta(2)AR: IR oligomer formation, likely comprising heteromers of homodimers.",
journal = "PLOS One",
title = "Demonstration of a Direct Interaction between beta(2)-Adrenergic Receptor and Insulin Receptor by BRET and Bioinformatics",
volume = "9",
number = "11",
doi = "10.1371/journal.pone.0112664"
}

Mandić, M., Drinovec, L., Glišić, S., Veljković, N. V., Nohr, J.,& Vrecl, M.. (2014). Demonstration of a Direct Interaction between beta(2)-Adrenergic Receptor and Insulin Receptor by BRET and Bioinformatics. in PLOS One, 9(11).
https://doi.org/10.1371/journal.pone.0112664

Mandić M, Drinovec L, Glišić S, Veljković NV, Nohr J, Vrecl M. Demonstration of a Direct Interaction between beta(2)-Adrenergic Receptor and Insulin Receptor by BRET and Bioinformatics. in PLOS One. 2014;9(11).
doi:10.1371/journal.pone.0112664 .

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