FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3124570178> ?p ?o ?g. }

• W3124570178 endingPage "100329" @default.
• W3124570178 startingPage "100329" @default.
• W3124570178 abstract "Recent functional and proteomic studies in eukaryotes (www.openprot.org) predict the translation of alternative open reading frames (AltORFs) in mature G-protein-coupled receptor (GPCR) mRNAs, including that of bradykinin B2 receptor (B2R). Our main objective was to determine the implication of a newly discovered AltORF resulting protein, termed AltB2R, in the known signaling properties of B2R using complementary methodological approaches. When ectopically expressed in HeLa cells, AltB2R presented predominant punctate cytoplasmic/perinuclear distribution and apparent cointeraction with B2R at plasma and endosomal/vesicular membranes. The presence of AltB2R increases intracellular [Ca2+] and ERK1/2-MAPK activation (via phosphorylation) following B2R stimulation. Moreover, HEK293A cells expressing mutant B2R lacking concomitant expression of AltB2R displayed significantly decreased maximal responses in agonist-stimulated Gαq-Gαi2/3–protein coupling, IP3 generation, and ERK1/2-MAPK activation as compared with wild-type controls. Conversely, there was no difference in cell-surface density as well as ligand-binding properties of B2R and in efficiencies of cognate agonists at promoting B2R internalization and β-arrestin 2 recruitment. Importantly, both AltB2R and B2R proteins were overexpressed in prostate and breast cancers, compared with their normal counterparts suggesting new associative roles of AltB2R in these diseases. Our study shows that BDKRB2 is a dual-coding gene and identifies AltB2R as a novel positive modulator of some B2R signaling pathways. More broadly, it also supports a new, unexpected alternative proteome for GPCRs, which opens new frontiers in fields of GPCR biology, diseases, and drug discovery. Recent functional and proteomic studies in eukaryotes (www.openprot.org) predict the translation of alternative open reading frames (AltORFs) in mature G-protein-coupled receptor (GPCR) mRNAs, including that of bradykinin B2 receptor (B2R). Our main objective was to determine the implication of a newly discovered AltORF resulting protein, termed AltB2R, in the known signaling properties of B2R using complementary methodological approaches. When ectopically expressed in HeLa cells, AltB2R presented predominant punctate cytoplasmic/perinuclear distribution and apparent cointeraction with B2R at plasma and endosomal/vesicular membranes. The presence of AltB2R increases intracellular [Ca2+] and ERK1/2-MAPK activation (via phosphorylation) following B2R stimulation. Moreover, HEK293A cells expressing mutant B2R lacking concomitant expression of AltB2R displayed significantly decreased maximal responses in agonist-stimulated Gαq-Gαi2/3–protein coupling, IP3 generation, and ERK1/2-MAPK activation as compared with wild-type controls. Conversely, there was no difference in cell-surface density as well as ligand-binding properties of B2R and in efficiencies of cognate agonists at promoting B2R internalization and β-arrestin 2 recruitment. Importantly, both AltB2R and B2R proteins were overexpressed in prostate and breast cancers, compared with their normal counterparts suggesting new associative roles of AltB2R in these diseases. Our study shows that BDKRB2 is a dual-coding gene and identifies AltB2R as a novel positive modulator of some B2R signaling pathways. More broadly, it also supports a new, unexpected alternative proteome for GPCRs, which opens new frontiers in fields of GPCR biology, diseases, and drug discovery. Until recently, it was believed that overlapping open reading frames (ORFs) and polycistronic mRNAs only occurred in prokaryotes and viruses. However, recent studies have challenged the notion of an mRNA coding for a single annotated protein and showed the pervasive properties of the translation machinery (1Mouilleron H. Delcourt V. Roucou X. Death of a dogma: Eukaryotic mRNAs can code for more than one protein.Nucleic Acids Res. 2016; 44: 14-23Crossref PubMed Scopus (45) Google Scholar). Multiple examples of mRNAs coding for an annotated coding sequence (RefORF) and an alternative open reading frame (AltORF) have been published in recent years (2Kondo T. Hashimoto Y. Kato K. Inagaki S. Hayashi S. Kageyama Y. Small peptide regulators of actin-based cell morphogenesis encoded by a polycistronic mRNA.Nat. Cell Biol. 2007; 9: 660-665Crossref PubMed Scopus (167) Google Scholar, 3Vanderperre B. Staskevicius A.B. Tremblay G. McCoy M. O’Neill M.A. Cashman N.R. Roucou X. An overlapping reading frame in the PRNP gene encodes a novel polypeptide distinct from the prion protein.FASEB J. 2011; 25: 2373-2386Crossref PubMed Scopus (33) Google Scholar, 4Bergeron D. Lapointe C. Bissonnette C. Tremblay G. Motard J. Roucou X. An out-of-frame overlapping reading frame in the ataxin-1 coding sequence encodes a novel ataxin-1 interacting protein.J. Biol. Chem. 2013; 288: 21824-21835Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 5Delcourt V. Franck J. Leblanc E. Narducci F. Robin Y.-M. Gimeno J.-P. Quanico J. Wisztorski M. Kobeissy F. Jacques J.-F. Roucou X. Salzet M. Fournier I. Combined mass spectrometry imaging and top-down microproteomics reveals evidence of a hidden proteome in ovarian cancer.EBioMedicine. 2017; 21: 55-64Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 6Samandi S. Roy A.V. Delcourt V. Lucier J.-F. Gagnon J. Beaudoin M.C. Vanderperre B. Breton M.-A. Motard J. Jacques J.-F. Brunelle M. Gagnon-Arsenault I. Fournier I. Ouangraoua A. Hunting D.J. et al.Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins.eLife. 2017; 6e27860Crossref PubMed Scopus (47) Google Scholar, 7Lee C. Lai H.-L. Lee Y.-C. Chien C.-L. Chern Y. The A2A adenosine receptor is a dual coding gene: A novel mechanism of gene usage and signal transduction.J. Biol. Chem. 2014; 289: 1257-1270Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 8Vanderperre B. Lucier J.-F. Bissonnette C. Motard J. Tremblay G. Vanderperre S. Wisztorski M. Salzet M. Boisvert F.-M. Roucou X. Direct detection of alternative open reading frames translation products in human significantly expands the proteome.PLoS One. 2013; 8e70698Crossref PubMed Scopus (111) Google Scholar, 9Akimoto C. Sakashita E. Kasashima K. Kuroiwa K. Tominaga K. Hamamoto T. Endo H. Translational repression of the McKusick–Kaufman syndrome transcript by unique upstream open reading frames encoding mitochondrial proteins with alternative polyadenylation sites.Biochim. Biophys. Acta. 2013; 1830: 2728-2738Crossref PubMed Scopus (20) Google Scholar, 10Ouelle D.E. Zindy F. Ashmun R.A. Sherr C.J. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest.Cell. 1995; 83: 993-1000Abstract Full Text PDF PubMed Scopus (1275) Google Scholar, 11Abramowitz J. Grenet D. Birnbaumer M. Torres H.N. Birnbaumer L. XLαs, the extra-long form of the α-subunit of the Gs G protein, is significantly longer than suspected, and so is its companion Alex.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8366-8371Crossref PubMed Scopus (52) Google Scholar, 12Chalick M. Jacobi O. Pichinuk E. Garbar C. Bensussan A. Meeker A. Ziv R. Zehavi T. Smorodinsky N.I. Hilkens J. Hanisch F.-G. Rubinstein D.B. Wreschner D.H. MUC1-ARF—a novel MUC1 protein that resides in the nucleus and is expressed by alternate reading frame translation of MUC1 mRNA.PLoS One. 2016; 11e0165031Crossref PubMed Scopus (7) Google Scholar, 13Autio K.J. Kastaniotis A.J. Pospiech H. Miinalainen I.J. Schonauer M.S. Dieckmann C.L. Hiltunen J.K. An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing.FASEB J. 2007; 22: 569-578Crossref PubMed Scopus (41) Google Scholar, 14Liu J. Yosten G.L.C. Ji H. Zhang D. Zheng W. Speth R.C. Samson W.K. Sandberg K. Selective inhibition of angiotensin receptor signaling through Erk1/2 pathway by a novel peptide.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2014; 306: R619-R626Crossref PubMed Scopus (14) Google Scholar, 15Klemke M. Kehlenbach R.H. Huttner W.B. Two overlapping reading frames in a single exon encode interacting proteins—a novel way of gene usage.EMBO J. 2001; 20: 3849-3860Crossref PubMed Scopus (96) Google Scholar), such as uMKKS1-2 (9Akimoto C. Sakashita E. Kasashima K. Kuroiwa K. Tominaga K. Hamamoto T. Endo H. Translational repression of the McKusick–Kaufman syndrome transcript by unique upstream open reading frames encoding mitochondrial proteins with alternative polyadenylation sites.Biochim. Biophys. Acta. 2013; 1830: 2728-2738Crossref PubMed Scopus (20) Google Scholar), AltMiD51 (6Samandi S. Roy A.V. Delcourt V. Lucier J.-F. Gagnon J. Beaudoin M.C. Vanderperre B. Breton M.-A. Motard J. Jacques J.-F. Brunelle M. Gagnon-Arsenault I. Fournier I. Ouangraoua A. Hunting D.J. et al.Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins.eLife. 2017; 6e27860Crossref PubMed Scopus (47) Google Scholar), AltPrP (3Vanderperre B. Staskevicius A.B. Tremblay G. McCoy M. O’Neill M.A. Cashman N.R. Roucou X. An overlapping reading frame in the PRNP gene encodes a novel polypeptide distinct from the prion protein.FASEB J. 2011; 25: 2373-2386Crossref PubMed Scopus (33) Google Scholar), AltATXN1 (4Bergeron D. Lapointe C. Bissonnette C. Tremblay G. Motard J. Roucou X. An out-of-frame overlapping reading frame in the ataxin-1 coding sequence encodes a novel ataxin-1 interacting protein.J. Biol. Chem. 2013; 288: 21824-21835Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), AltHNRNPUL1 (8Vanderperre B. Lucier J.-F. Bissonnette C. Motard J. Tremblay G. Vanderperre S. Wisztorski M. Salzet M. Boisvert F.-M. Roucou X. Direct detection of alternative open reading frames translation products in human significantly expands the proteome.PLoS One. 2013; 8e70698Crossref PubMed Scopus (111) Google Scholar), ARF (10Ouelle D.E. Zindy F. Ashmun R.A. Sherr C.J. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest.Cell. 1995; 83: 993-1000Abstract Full Text PDF PubMed Scopus (1275) Google Scholar), ALEX (11Abramowitz J. Grenet D. Birnbaumer M. Torres H.N. Birnbaumer L. XLαs, the extra-long form of the α-subunit of the Gs G protein, is significantly longer than suspected, and so is its companion Alex.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8366-8371Crossref PubMed Scopus (52) Google Scholar, 15Klemke M. Kehlenbach R.H. Huttner W.B. Two overlapping reading frames in a single exon encode interacting proteins—a novel way of gene usage.EMBO J. 2001; 20: 3849-3860Crossref PubMed Scopus (96) Google Scholar), MUC1-ARF (12Chalick M. Jacobi O. Pichinuk E. Garbar C. Bensussan A. Meeker A. Ziv R. Zehavi T. Smorodinsky N.I. Hilkens J. Hanisch F.-G. Rubinstein D.B. Wreschner D.H. MUC1-ARF—a novel MUC1 protein that resides in the nucleus and is expressed by alternate reading frame translation of MUC1 mRNA.PLoS One. 2016; 11e0165031Crossref PubMed Scopus (7) Google Scholar), AltRPP14 (13Autio K.J. Kastaniotis A.J. Pospiech H. Miinalainen I.J. Schonauer M.S. Dieckmann C.L. Hiltunen J.K. An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing.FASEB J. 2007; 22: 569-578Crossref PubMed Scopus (41) Google Scholar). About 183,000 AltORFs are predicted in the human transcriptome, of which 76% are predicted to be encoded in coding RNAs (6Samandi S. Roy A.V. Delcourt V. Lucier J.-F. Gagnon J. Beaudoin M.C. Vanderperre B. Breton M.-A. Motard J. Jacques J.-F. Brunelle M. Gagnon-Arsenault I. Fournier I. Ouangraoua A. Hunting D.J. et al.Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins.eLife. 2017; 6e27860Crossref PubMed Scopus (47) Google Scholar), the remainder being predicted to be encoded on RNAs previously annotated as noncoding. These AltORFs localize in the 5'/3'-UTRs or overlap the annotated coding sequence (CDS) in the +2 or the +3 reading frames. Several specific examples have been reported in recent years, the first one being ALEX, an AltORF encoded in the coding sequence of the G protein XLαs (11Abramowitz J. Grenet D. Birnbaumer M. Torres H.N. Birnbaumer L. XLαs, the extra-long form of the α-subunit of the Gs G protein, is significantly longer than suspected, and so is its companion Alex.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8366-8371Crossref PubMed Scopus (52) Google Scholar, 15Klemke M. Kehlenbach R.H. Huttner W.B. Two overlapping reading frames in a single exon encode interacting proteins—a novel way of gene usage.EMBO J. 2001; 20: 3849-3860Crossref PubMed Scopus (96) Google Scholar). ALEX interacts with XLαs and prevents it from activating the adenylate cyclase. More recently, the rat Adora2a gene has been shown to code for the adenosine A2a receptor and a novel alternative protein encoded in an AltORF located in the 5’UTR, named uORF5. Expression of uORF5 modulates the expression of several genes involved in the mitogen-activated protein kinases (MAPK)/ERK pathway, and stimulation of the A2aR receptor results in an increase in uORF5 expression (7Lee C. Lai H.-L. Lee Y.-C. Chien C.-L. Chern Y. The A2A adenosine receptor is a dual coding gene: A novel mechanism of gene usage and signal transduction.J. Biol. Chem. 2014; 289: 1257-1270Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Another recent example is the discovery of the seven amino acid peptide named PEP7, which is encoded in an AltORF present in the 5'UTR of the rat angiotensin AT1aR gene (14Liu J. Yosten G.L.C. Ji H. Zhang D. Zheng W. Speth R.C. Samson W.K. Sandberg K. Selective inhibition of angiotensin receptor signaling through Erk1/2 pathway by a novel peptide.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2014; 306: R619-R626Crossref PubMed Scopus (14) Google Scholar). Exogenously applied PEP7 inhibited β-arrestin-dependent MAPK activation in HEK293 cells expressing AT1aR. In vivo, PEP7 blocked angiotensin II-stimulated saline intake (14Liu J. Yosten G.L.C. Ji H. Zhang D. Zheng W. Speth R.C. Samson W.K. Sandberg K. Selective inhibition of angiotensin receptor signaling through Erk1/2 pathway by a novel peptide.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2014; 306: R619-R626Crossref PubMed Scopus (14) Google Scholar). Using the Openprot database (16Brunet M.A. Brunelle M. Lucier J.-F. Delcourt V. Levesque M. Grenier F. Samandi S. Leblanc S. Aguilar J.-D. Dufour P. Jacques J.-F. Fournier I. Ouangraoua A. Scott M.S. Boisvert F.-M. et al.OpenProt: A more comprehensive guide to explore eukaryotic coding potential and proteomes.Nucleic Acids Res. 2019; 47: D403-D410PubMed Google Scholar), we identified a total of 4645 putative novel AltORFs with a minimum length of 30 codons in 81% of the 830 human G-protein-coupled receptor (GPCR) genes (Fig. 1A; see Table S1 for complete list of AltORFs). In total, 42% of them are localized in the 3'UTR, 23% in the 5'UTR, and more than 35% are predicted to overlap the annotated GPCR coding sequence. The human BDKRB2 gene has nine predicted AltORFs (Fig. 1B, Table S2). One of them is a 474 nucleotide AltORF overlapping the coding sequence of B2R in the +3 reading frame (Fig. 1B). This AltORF, which we termed AltB2R, is predicted to produce a 157 amino acid long protein with a 17 kDa molecular weight and an isoelectric point of 6.58 (ProtParam tool, ExPASy server). PSORT bioinformatic analysis indicates that AltB2R is likely to be a soluble cytosolic protein rather than a transmembrane protein (not shown). B2R exhibits constitutive expression in various tissues/organs and exerts pleiotropic (beneficial and deleterious) effects in various physiopathological conditions, such as diabetes, cardiovascular diseases, and cancers (17Regoli D. Nsa Allogho S. Rizzi A. Gobeil F.J. Bradykinin receptors and their antagonists.Eur. J. Pharmacol. 1998; 348: 1-10Crossref PubMed Scopus (219) Google Scholar, 18Couture R. Blaes N. Girolami J.-P. Kinin receptors in vascular biology and pathology.Curr. Vasc. Pharmacol. 2014; 12: 223-248Crossref PubMed Scopus (46) Google Scholar, 19Regoli D. Plante G.E. Gobeil F. Impact of kinins in the treatment of cardiovascular diseases.Pharmacol. Ther. 2012; 135: 94-111Crossref PubMed Scopus (64) Google Scholar, 20Figueroa C.D. Ehrenfeld P. Bhoola K.D. Kinin receptors as targets for cancer therapy.Expert Opin. Ther. Targets. 2012; 16: 299-312Crossref PubMed Scopus (30) Google Scholar, 21da Costa P.L.N. Sirois P. Tannock I.F. Chammas R. The role of kinin receptors in cancer and therapeutic opportunities.Cancer Lett. 2014; 345: 27-38Crossref PubMed Scopus (65) Google Scholar). Its principal peptide agonist ligands are the nona- and decapeptide bradykinin and Lys-BK (alias kallidin; KD), respectively. Whereas the carboxy-terminally truncated peptide metabolites desArg9-BK and desArg10-KD, derived from enzymatic activities of carboxypeptidases M/N, selectively bind to the other type of kinin receptor, the inducible B1R (22Leeb-Lundberg L.M.F. International union of pharmacology. XLV. Classification of the kinin receptor family: From molecular mechanisms to pathophysiological consequences.Pharmacol. Rev. 2005; 57: 27-77Crossref PubMed Scopus (757) Google Scholar). B2R can initiate multiple signaling pathways through multiple G proteins (e.g., Gαq/11, Gα12/13, Gαi and Gαs) and β-Arrestins (βarrs) in mammalian cells (22Leeb-Lundberg L.M.F. International union of pharmacology. XLV. Classification of the kinin receptor family: From molecular mechanisms to pathophysiological consequences.Pharmacol. Rev. 2005; 57: 27-77Crossref PubMed Scopus (757) Google Scholar, 23Shenoy S.K. Lefkowitz R.J. Receptor regulation: β-arrestin moves up a notch.Nat. Cell Biol. 2005; 7: 1059-1061Crossref Scopus (17) Google Scholar, 24Leschner J. Wennerberg G. Feierler J. Bermudez M. Welte B. Kalatskaya I. Wolber G. Faussner A. Interruption of the ionic lock in the bradykinin B2 receptor results in constitutive internalization and turns several antagonists into strong agonists.J. Pharmacol. Exp. Ther. 2013; 344: 85-95Crossref PubMed Scopus (13) Google Scholar). These latter are often linked to successive activation of MAPK/ERK1/2 involved in transcription of genes that promote cellular proliferation and survival (22Leeb-Lundberg L.M.F. International union of pharmacology. XLV. Classification of the kinin receptor family: From molecular mechanisms to pathophysiological consequences.Pharmacol. Rev. 2005; 57: 27-77Crossref PubMed Scopus (757) Google Scholar, 25Wang G. Sun J. Liu G. Fu Y. Zhang X. Bradykinin promotes cell proliferation, migration, invasion, and tumor growth of gastric cancer through ERK signaling pathway: Role of bradykinin in GC.J. Cell. Biochem. 2017; 118: 4444-4453Crossref PubMed Scopus (13) Google Scholar, 26Khoury E. Nikolajev L. Simaan M. Namkung Y. Laporte S.A. Differential regulation of endosomal GPCR/β-arrestin complexes and trafficking by MAPK.J. Biol. Chem. 2014; 289: 23302-23317Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In this study, we investigated whether the expression of AltB2R can directly regulate B2R activity. Our results demonstrated that AltB2R can interact with B2R protein and enhance B2R-mediated transduction signals occurring through Gαq/i2/i3 signaling activity. We also showed bona fide endogenous coexpression of AltB2R and B2R with variable expression patterns, in clinical specimens of different types of solid human cancers, indicating that it is likely to perform important functions in these diseases. To test whether AltB2R is coexpressed with B2R, an HA tag was introduced in frame with AltB2R to produce carboxy-tagged AltB2R (AltB2RHA), within the B2R coding sequence. Since B2R has a C-terminal Flag tag, this construct is termed B2R(HA)Flag to indicate that the HA tag is silent within the B2R reading frame (Fig. 2A). Control constructs included B2RFlag and AltB2RHA. B2RFlag and AltB2RHA were detected in cells transfected with B2R(HA)Flag, indicating that both proteins are expressed from the coding sequence of B2R (Fig. 2B). Immunofluorescence staining in permeabilized HeLa cells transfected with AltB2RHA showed the cytoplasmic localization of the protein with a nonhomogeneous distribution (Fig. 2C). Cotransfection with B2RFlag indicated that both proteins colocalized at the cell membrane, perinuclear regions, and in cytoplasmic foci, most likely to be endosome and/or plasma membrane transport vesicles (Fig. 2C). Similar experiments conducted in nonpermeabilized cell conditions gave strong punctate surface staining of the B2R (Fig. 2D). Further confirmation of the cytoplasmic localization of AltB2R was obtained by western blot analysis of highly purified subcellular fractions (nuclei, cytoplasm, and cell membrane fractions) (Fig. 2E). Cytoplasmic colocalization of B2R and AltB2R suggested a possible interaction between both proteins. To test this hypothesis, we used bimolecular fluorescence complementation (BiFC) based on the association of split VENUS fragments composed of VN (amino acids 1–210) and VC (amino acids 210–238). B2RFlag-VN and VC-AltB2RHA displayed a membranous/cytoplasmic localization (Fig. 3A), in accordance with our previous microscopy and western blot data. Cells cotransfected with B2RFlag-VN and VC-AltB2RHA displayed Venus fluorescence, confirming B2R/AltB2R interaction in cellulo (Fig. 3A and Fig. S3B). In support of a specific interaction between B2R and AltB2R, competition with B2R lacking the VN fragment or AltB2R lacking the VC fragment completely prevented the formation of BiFC complexes (Fig. 3A). Further experiments performed with constructs where the Venus fragments were switched around to B2RFlag-VC and VN-AltB2RHA gave similar positive results (data not shown). In control experiments, cells expressing either VN or VC, VN and VC, B2R-VN and VC, or VC-AltB2R and VN did not display any BiFC signal (Fig. 3B). To demonstrate the specificity of interaction between AltB2R and B2R, we conducted similar experiments in cells transfected with the human B1RFlag-VC and VN-AltB2RHA. In this experimental setting, no BiFC signal could be detected between the two proteins, suggesting some degree of specificity in the association between AltB2R and B2R (Fig. S3A). Next, we tested if the physical interaction between AltB2R and B2R could modulate B2R signaling processes. Considering that many signaling pathways of B2R converge toward the activation of MAPK ERK1/2 (27Blaukat A. Structure and signalling pathways of kinin receptors.Andrologia. 2003; 35: 17-23Crossref PubMed Scopus (54) Google Scholar), we first studied the level of phosphorylation of ERK1/2 upon agonist stimulation of endogenous B2R in HeLa cells. The metabolically stabilized, selective, potent B2R agonists NG291 and RMP-7 were used in these functional assays and in others to avoid possible interference and unpredictable actions of peptidases on B2R signaling (ex. formation of active metabolites at receptors other than B2R, cross talk between peptidases and B2R) in the cell models employed for the study, i.e., HeLa and HEK293 cells (28Bausch-Fluck D. Hofmann A. Bock T. Frei A.P. Cerciello F. Jacobs A. Moest H. Omasits U. Gundry R.L. Yoon C. Schiess R. Schmidt A. Mirkowska P. Härtlová A. Van Eyk J.E. et al.A mass spectrometric-derived cell surface protein atlas.PLoS One. 2015; 10e0121314Crossref PubMed Scopus (158) Google Scholar, 29Campbell D.J. Neprilysin inhibitors and bradykinin.Front. Med. 2018; 5: 257Crossref Scopus (24) Google Scholar). In mock-HeLa cells stimulated with RMP-7, levels of phospho-ERK1/2 reached a maximum 10 min poststimulation and returned to basal levels after 30 min (Fig. 4A). ERK1/2 phosphorylation kinetics were similar in HeLa cells stably expressing AltB2R, but maximum levels at 10 min were significantly higher compared with mock cells (Fig. 4A). We performed the same assays using instead the biostable, selective B1R agonist NG29 (30Savard M. Côté J. Tremblay L. Neugebauer W. Regoli D. Gariépy S. Hébert N. Gobeil F. Safety and pharmacokinetics of a kinin B1 receptor peptide agonist produced with different counter-ions.Biol. Chem. 2016; 397: 365-372Crossref PubMed Scopus (1) Google Scholar) and did not observe any difference in the increased levels of phosphorylated ERK1/2 between mock and AltB2RFlag cells (Fig. S3), indicating some functional specificity of AltB2R toward the modulation of MAPK from B2R. Since B2R-induced MAPK ERK1/2 activation often requires the mobilization of intracellular calcium (i[Ca2+]), we then analyzed whether the presence of AltB2R can also impact intensity of intracellular Ca2+ mobilization in B2R-stimulated cells previously loaded with the fluorescent Ca2+-sensitive dye Fluo-4. In mock stable cells, RMP-7 treatment induced a significant increase in i[Ca2+] (Fig. 4B). In AltB2RFlag-expressing cells, basal levels of free Ca2+ were significantly lower compared with mock cells. Interestingly, these latter cells had significantly higher Ca2+ mobilization levels compared with mock cells in response to RMP-7 (Fig. 4B). From these observations, it could be inferred that the potentiation of B2R agonist responses elicited by AltB2R may involve upstream targets of MAPK/ERK and Ca2+ pathways. In order to further substantiate the magnitude of effects of AltB2R on the pharmacological and signaling properties of B2R, we generated a mutant construct termed B2R(Ø), expressed in HEK293A cells (Fig. S1A). This construct is a monocistronic version of B2R with the mutation C168T. In this mutant, a premature stop codon is introduced in the AltB2R coding sequence (Q11stop) in a manner synonymous for B2R (L56 L). Thus, the B2R encodes B2R and AltB2R, whereas mutant B2R(Ø) encodes only B2R. Indeed, western blotting showed that cells transfected with B2R(Ø) did not express AltB2R, while having relatively unaltered B2R expression compared with mutant cells encoding both B2R/AtlB2R (Fig. S1B). Western blotting experiments showed that in B2R-expressing cells stimulated with RMP-7, levels of phospho-ERK1/2 reached a maximum at 5 min poststimulation and returned to basal levels after 15 min (Fig. 5A). ERK1/2 phosphorylation kinetics were similar in cells transfected with B2R(Ø), but maximal levels of phospho-ERK1/2 were significantly lower (Fig. 5A). To consolidate the western blot data, AlphaLISA assays were also conducted in parallel in the same cell lines for the quantitative analyses of B2R-stimulated ERK1/2 phosphorylation (Fig. 5, B and C). Again, the results showed decreased maximal levels of phospho-ERK1/2 in B2R(Ø) versus B2R-transfected cells when stimulated with either RMP-7 or NG291. Since calcium mobilization precedes ERK1/2 phosphorylation and results from 1,4,5-inositol trisphosphate (IP3) binding to IP3R at the endoplasmic reticulum membrane, we tested whether the presence AltB2R could also amplify production of IP3 resulting from B2R activation. IP3 formation (indirectly measured via its stabilized metabolite IP1) was assessed following generation of full concentration-response profiles of B2R agonists RMP-7 and NG291 in B2R and B2R(Ø)-transfected cells. Results indicated no significant difference in potencies (EC50 values) of agonists between B2R- and B2R(Ø)-transfected cells (Fig. 5, D and E). However, significantly lower agonist efficacies (Emax values) were noted in cells without AltB2R expression (16 and 22% reduction of efficacy for RMP-7 and NG291, respectively). These results indicate that AltB2R may impact B2R signal transduction pathways at various levels. The observation that AltB2R is necessary to achieve a full production of IP3 upon B2R stimulation suggests that AltB2R modulates coupling between B2R and Gαq and/or Gαi. To test this hypothesis, we used live-cell bioluminescence resonance energy transfer (BRET) measurements to monitor the activation of G proteins by B2R. HEK293A cells were cotransfected with vectors coding for Gαq-RlucII or Gαi2/3-RlucII, Gβ, GFP10-Gγ1, and B2R or B2R(Ø). In this experimental setting, the dissociation of Gαq-RlucII or Gαi2/3-RlucII from GFP10-Gγ1 results in a decrease of BRET signals. BRET data in Figure 6, A–C are presented relative to the maximum B2R-induced BRET response. We observed no difference of the EC50 for the dissociation Gαq/Gγ1 in B2R-transfected cells compared with B2R(Ø)-transfected cells (Fig. 6A). There was a significant decrease in the maximal dissociation Emax (100 ± 10% versus 74 ± 8% for B2R and B2R(Ø), respectively). For the dissociation Gαi2/Gγ1, we observed no difference in the EC50 whether AltB2R was present or absent, but Emax decreased from 100 ± 11% in B2R-transfected cells to 66 ± 12% in B2R(Ø)-transfected cells (Fig. 6B). For the dissociation of Gαi3/Gγ1, the EC50 was similar in B2R- and B2R(Ø)-transfected cells, but Emax was significantly lower in B2R(Ø)-transfected cells (60 ± 22% versus 100 ± 21%) (Fig. 6C). Since both G proteins and β-arrestins commonly participate in activation of MAPK pathways, we extended the BRET analysis to determine the effects of AltB2R on the efficacy of β-arrestin2 recruitment at the plasma membrane of HEK293A cells transfected with vectors coding for β-arrestin2-RLucII, rGFP-CAAX, and B2R or B2R(Ø). BRET signals were monitored after addition of NG291. We detected no difference in the recruitment of β-arrestin2 in cells either expressing or not AltB2R (Fig. 6D); similar results were obtained with BK (data not shown). Moreover, AltB2R did not impair both the magnitude and rate of agonist-promoted internalization of B2R, most likely dependent on β-arrestin2 (31Zimmerman B. Simaan M. Akoume M.-Y. Houri N. Chevallier S. Séguéla P. Laporte S.A. Role of ßarrestins in bradykinin B2 receptor-mediated signalling.Cell. Signal. 2011; 23: 648-659Crossref PubMed Scopus (31) Google Scholar) (Fig. 6, E and F). Hence, these results suggest" @default.
• W3124570178 created "2021-02-01" @default.
• W3124570178 creator A5000413143 @default.
• W3124570178 creator A5012450160 @default.
• W3124570178 creator A5026031535 @default.
• W3124570178 creator A5033478323 @default.
• W3124570178 creator A5040609487 @default.
• W3124570178 creator A5053182265 @default.
• W3124570178 creator A5065437733 @default.
• W3124570178 date "2021-01-01" @default.
• W3124570178 modified "2023-10-11" @default.
• W3124570178 title "Potentiation of B2 receptor signaling by AltB2R, a newly identified alternative protein encoded in the human bradykinin B2 receptor gene" @default.
• W3124570178 cites W1970569349 @default.
• W3124570178 cites W1974041902 @default.
• W3124570178 cites W1980587229 @default.
• W3124570178 cites W1985255881 @default.
• W3124570178 cites W1987324997 @default.
• W3124570178 cites W1987704983 @default.
• W3124570178 cites W2000605942 @default.
• W3124570178 cites W2010075485 @default.
• W3124570178 cites W2010690249 @default.
• W3124570178 cites W2025383208 @default.
• W3124570178 cites W2026433130 @default.
• W3124570178 cites W2027875197 @default.
• W3124570178 cites W2029173942 @default.
• W3124570178 cites W2040415319 @default.
• W3124570178 cites W2048657452 @default.
• W3124570178 cites W2053333991 @default.
• W3124570178 cites W2058401979 @default.
• W3124570178 cites W2058937802 @default.
• W3124570178 cites W2059143988 @default.
• W3124570178 cites W2059496903 @default.
• W3124570178 cites W2062964643 @default.
• W3124570178 cites W2067700675 @default.
• W3124570178 cites W2078266900 @default.
• W3124570178 cites W2084891010 @default.
• W3124570178 cites W2084955416 @default.
• W3124570178 cites W2085220756 @default.
• W3124570178 cites W2088883717 @default.
• W3124570178 cites W2090131666 @default.
• W3124570178 cites W2091588586 @default.
• W3124570178 cites W2092247923 @default.
• W3124570178 cites W2094610765 @default.
• W3124570178 cites W2101718405 @default.
• W3124570178 cites W2115565372 @default.
• W3124570178 cites W2149661661 @default.
• W3124570178 cites W2179178090 @default.
• W3124570178 cites W2192733079 @default.
• W3124570178 cites W2413810829 @default.
• W3124570178 cites W2469309996 @default.
• W3124570178 cites W2517932401 @default.
• W3124570178 cites W2536537637 @default.
• W3124570178 cites W2559801043 @default.
• W3124570178 cites W2559953833 @default.
• W3124570178 cites W2581290476 @default.
• W3124570178 cites W2610959580 @default.
• W3124570178 cites W2620890793 @default.
• W3124570178 cites W2734798616 @default.
• W3124570178 cites W2765153686 @default.
• W3124570178 cites W2766574082 @default.
• W3124570178 cites W2770089238 @default.
• W3124570178 cites W2782030629 @default.
• W3124570178 cites W2795785060 @default.
• W3124570178 cites W2801240764 @default.
• W3124570178 cites W2889990268 @default.
• W3124570178 cites W2890618895 @default.
• W3124570178 cites W2971297443 @default.
• W3124570178 cites W2981982826 @default.
• W3124570178 cites W2996446320 @default.
• W3124570178 cites W4246857619 @default.
• W3124570178 doi "https://doi.org/10.1016/j.jbc.2021.100329" @default.
• W3124570178 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7949122" @default.
• W3124570178 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/33497625" @default.
• W3124570178 hasPublicationYear "2021" @default.
• W3124570178 type Work @default.
• W3124570178 sameAs 3124570178 @default.
• W3124570178 citedByCount "9" @default.
• W3124570178 countsByYear W31245701782021 @default.
• W3124570178 countsByYear W31245701782022 @default.
• W3124570178 countsByYear W31245701782023 @default.
• W3124570178 crossrefType "journal-article" @default.
• W3124570178 hasAuthorship W3124570178A5000413143 @default.
• W3124570178 hasAuthorship W3124570178A5012450160 @default.
• W3124570178 hasAuthorship W3124570178A5026031535 @default.
• W3124570178 hasAuthorship W3124570178A5033478323 @default.
• W3124570178 hasAuthorship W3124570178A5040609487 @default.
• W3124570178 hasAuthorship W3124570178A5053182265 @default.
• W3124570178 hasAuthorship W3124570178A5065437733 @default.
• W3124570178 hasBestOaLocation W31245701781 @default.
• W3124570178 hasConcept C104317684 @default.
• W3124570178 hasConcept C153939273 @default.
• W3124570178 hasConcept C170493617 @default.
• W3124570178 hasConcept C185592680 @default.
• W3124570178 hasConcept C25274449 @default.
• W3124570178 hasConcept C2779591629 @default.
• W3124570178 hasConcept C54355233 @default.
• W3124570178 hasConcept C62478195 @default.
• W3124570178 hasConcept C86803240 @default.

• next