title: Dopamine-2 receptor extracellular N-terminus regulates receptor surface availability and is the target of human pathogenic antibodies from children with movement and psychiatric disorders.
abstract: Anti-Dopamine-2 receptor (D2R) antibodies have been recently identified in a subgroup of children with autoimmune movement and psychiatric disorders, however the epitope(s) and mechanism of pathogenicity remain unknown. Here we report a major biological role for D2R extracellular N-terminus as a regulator of receptor surface availability, and as a major epitope targeted and impaired in brain autoimmunity. In transfected human cells, purified anti-D2R antibody from patients specifically and significantly reduced human D2R surface levels. Next, human D2R mutants modified in their extracellular domains were subcloned, and we analyzed the region bound by 35 anti-D2R antibody-positive patient sera using quantitative flow cytometry on live transfected cells. We found that N-glycosylation at amino acids N5 and/or N17 was critical for high surface expression in interaction with the last 15 residues of extracellular D2R N-terminus. No anti-D2R antibody-positive patient sera bound to the three extracellular loops, but all patient sera (35/35) targeted the extracellular N-terminus. Overall, patient antibody binding was dependent on two main regions encompassing amino acids 20 to 29, and 23 to 37. Residues 20 to 29 contributed to the majority of binding (77%, 27/35), among which 26% (7/27) sera bound to amino acids R20, P21, and F22, 37% (10/27) patients were dependent on residues at positions 26 and 29, that are different between humans and mice, and 30% (8/27) sera required R20, P21, F22, N23, D26, and A29. Seven patient sera bound to the region 23 to 37 independently of D26 and A29, but most sera exhibited N-glycosylation-independent epitope recognition at N23. Interestingly, no evident segregation of binding pattern according to patient clinical phenotype was observed. D2R N-terminus is a central epitope in autoimmune movement and psychiatric disorders and this knowledge could help the design of novel specific immune therapies tailored to improve patient outcome.
authors.affiliation: Brain Autoimmunity group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Nese
authors.initials: N
authors.lastname: Sinmaz
authors.affiliation: Brain Autoimmunity group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Fiona
authors.initials: F
authors.lastname: Tea
authors.affiliation: Brain Autoimmunity group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Deepti
authors.initials: D
authors.lastname: Pilli
authors.affiliation: Brain Autoimmunity group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Alicia
authors.initials: A
authors.lastname: Zou
authors.affiliation: Brain Autoimmunity group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Mazen
authors.initials: M
authors.lastname: Amatoury
authors.affiliation: Brain Autoimmunity group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Tina
authors.initials: T
authors.lastname: Nguyen
authors.affiliation: Brain Autoimmunity group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Vera
authors.initials: V
authors.lastname: Merheb
authors.affiliation: Brain Autoimmunity group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Sudarshini
authors.initials: S
authors.lastname: Ramanathan
authors.affiliation: Membrane Repair group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Sandra T
authors.initials: ST
authors.lastname: Cooper
authors.affiliation: Brain Autoimmunity group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Russell C
authors.initials: RC
authors.lastname: Dale
authors.affiliation: Brain Autoimmunity group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, University of Sydney, Westmead, NSW, 2145, Australia. fabienne.brilot@sydney.edu.au.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Brilot
mesh_terms: D000293:Adolescent; D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D005110:Extracellular Space; D006031:Glycosylation; D057809:HEK293 Cells; D006624:Hippocampus / Q000276:immunology / Q000473:pathology; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007223:Infant; D001523:Mental Disorders / Q000276:immunology* / Q000473:pathology; D009069:Movement Disorders / Q000276:immunology* / Q000473:pathology; D009154:Mutation; D058953:Neural Stem Cells / Q000276:immunology / Q000473:pathology; D000072417:Protein Domains; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D001323:Autoantibodies; C581293:DRD2 protein, human; C000600618:DRD2 protein, mouse; D007074:Immunoglobulin G; D017448:Receptors, Dopamine D2
keywords: Autoantibody; Autoimmune movement and psychiatric disorders; Dopamine-2 receptor; Epitope; N-terminus; Pathogenicity