57005d966494e1874618381bed714b7f pmc_31936298.pdf 85d57640c604cba630917508c4e66e299f7d6dc3 pmc_31936298.pdf 4598cbe7db15e0b71b01724d97ac0d3b644948114f1e4897b43bb7a399c1c350 pmc_31936298.pdf Title: Expression of Melatonin and Dopamine D3 Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension Subject: Background: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D3 receptors (D3Rs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by D3R and either MT1 (MT1R) or MT2 (MT2R) melatonin receptors. Heteromerization led to the blockade of D3R-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. Conclusions: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of D3R–MT1R and D3R–MT1R was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2. Keywords: circadian rhythm; glaucoma; G-protein-coupled receptor GPCR heteromer; melatonin MT1 and MT2 receptors; G protein coupling; mitogen-activated protein kinase pathway (MAPK); label-free dynamic mass redistribution (DMR); retina; human 59HCE cells Author: Irene Reyes-Resina, Hanan Awad Alkozi, Anna del Ser-Badia, Juan Sánchez-Naves, Jaume Lillo, Jasmina Jiménez, Jesús Pintor, Gemma Navarro and Rafael Franco Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Jan 8 12:53:02 2020 CET ModDate: Wed Jan 8 12:53:02 2020 CET Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 20 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3395661 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- DIOJDM+URWPalladioL-Ital Type 1 Custom yes yes no 116 0 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ぉの〠こどぴふ〠ちのつ〠ぉの〠ざどぴひは〠ぐひはへどねどぴべ〠がどでちぴどはの〠ぁびびちべ〠〨ぐがぁ〩〠 Item: Title: いちぴち〠ぁのちぬべびどび〠 Item: Title: げづびふぬぴび〠 Children: Item: Title: いはばちねどのづ〠い〳〠げづっづばぴはひび〠ぉのぴづひちっぴ〠ぷどぴと〠きづぬちぴはのどの〠きご〱〠げづっづばぴはひび〠どの〠ぴとづ〠えぅか〭〲〹〳ご〠ぃづぬぬび〠 Item: Title: うふのっぴどはのちぬ〠ぃとちひちっぴづひどぺちぴどはの〠はて〠い〳〭きご〱〠えづぴづひはひづっづばぴはひ〠ぃはねばぬづへづび〠どの〠えぅか〭〲〹〳ご〠ぃづぬぬび〠 Item: Title: いはばちねどのづ〠い〳〠げづっづばぴはひび〠ぉのぴづひちっぴ〠ぷどぴと〠きづぬちぴはのどの〠きご〲〠げづっづばぴはひび〠どの〠えぅか〭〲〹〳ご〠ぃづぬぬび〠 Item: Title: うふのっぴどはのちぬ〠ぃとちひちっぴづひどぺちぴどはの〠はて〠ぴとづ〠い〳〭きご〲〠えづぴづひはひづっづばぴはひ〠ぃはねばぬづへづび〠どの〠えぅか〭〲〹〳ご〠ぃづぬぬび〠 Item: Title: いづぴづっぴどはの〠ちのつ〠うふのっぴどはのちぬ〠ぃとちひちっぴづひどぺちぴどはの〠はて〠い〳〭きご〱〠ちのつ〠い〳〭きご〲〠えづぴづひはひづっづばぴはひ〠ぃはねばぬづへづび〠どの〠えふねちの〠ぎはの〭ぐどでねづのぴづつ〠ぃどぬどちひべ〠あはつべ〠ぅばどぴとづぬどちぬ〠ぃづぬぬび〠 Item: Title: いどててづひづのぴどちぬ〠ぅへばひづびびどはの〠はて〠い〳〭きご〱〠ちのつ〠い〳〭きご〲〠えづぴづひはひづっづばぴはひ〠ぃはねばぬづへづび〠どの〠ぴとづ〠ぇぬちふっはねちぴはふび〠ぅべづ〠 Item: Title: いどびっふびびどはの〠 Item: Title: Item: Title: げづてづひづのっづび Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: ZXDKIP+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: ZXDKIP+EURM10 Flags: Symbolic 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Subtype: Link Rect: 100, 586, 137, 596 Annotation: Subtype: Link Rect: 143, 586, 179, 596 Annotation: Subtype: Link Rect: 342, 559, 380, 570 Annotation: Subtype: Link Rect: 386, 559, 422, 570 Annotation: Subtype: Link Rect: 100, 521, 137, 531 Annotation: Subtype: Link Rect: 143, 521, 179, 531 Annotation: Subtype: Link Rect: 453, 495, 491, 506 Annotation: Subtype: Link Rect: 366, 456, 403, 467 Annotation: Subtype: Link Rect: 76, 375, 166, 406 Annotation: Subtype: Link Rect: 234, 370, 407, 380 Checksum: 44a0a72d Type: CRC32 Checksum: 57005d966494e1874618381bed714b7f Type: MD5 Checksum: 85d57640c604cba630917508c4e66e299f7d6dc3 Type: SHA-1 Checksum: 4598cbe7db15e0b71b01724d97ac0d3b644948114f1e4897b43bb7a399c1c350 Type: SHA-256