title: Metabotropic glutamate receptor 3 (mGlu3; mGluR3; GRM3) in schizophrenia: Antibody characterisation and a semi-quantitative western blot study.
abstract: Metabotropic glutamate receptor 3 (mGlu3, mGluR3), encoded by GRM3, is a risk gene for schizophrenia and a therapeutic target. It is unclear whether expression of the receptor is altered in the disorder or related to GRM3 risk genotype. Antibodies used to date to assess mGlu3 in schizophrenia have not been well validated.
  
  
  
  To characterise six commercially available anti-mGlu3 antibodies for use in human brain, and then conduct a semi-quantitative study of mGlu3 immunoreactivity in schizophrenia.
  
  
  
  Antibodies tested using Grm3-/-and Grm2-/-/3-/-mice and transfected HEK293T/17 cells. Western blotting on membrane protein isolated from superior temporal cortex of 70 patients with schizophrenia and 87 healthy comparison subjects, genotyped for GRM3 SNP rs10234440.
  
  
  
  One (out of six) anti-mGlu3 antibodies was fully validated, a C-terminal antibody which detected monomeric (~100kDa) and dimeric (~200kDa) mGlu3. A second, N-terminal, antibody detected the 200kDa band but also produced non-specific bands. Using the C-terminal antibody for western blotting in human brain, mGlu3 immunoreactivity was found to decline with age, and was affected by pH and post mortem interval. There were no differences in monomeric or dimeric mGlu3 immunoreactivity in schizophrenia or in relation to GRM3 genotype. The antibody was not suitable for immunohistochemistry.
  
  
  
  These data highlight the value of knockout mouse tissue for antibody validation, and the need for careful antibody characterisation. The schizophrenia data show that involvement of GRM3 in the disorder and its genetic risk architecture is not reflected in total membrane mGlu3 immunoreactivity in superior temporal cortex.
authors.affiliation: Department of Psychiatry, University of Oxford, Oxford, United Kingdom.
authors.forename: Aintzane
authors.initials: A
authors.lastname: García-Bea
authors.affiliation: Department of Psychiatry, University of Oxford, Oxford, United Kingdom.
authors.forename: Mary A
authors.initials: MA
authors.lastname: Walker
authors.affiliation: Lieber Institute for Brain Development, Baltimore, USA; Department of Neurology, Johns Hopkins School of Medicine, Baltimore, USA; Department of Psychiatry & Behavioral Sciences, Johns Hopkins School of Medicine, Baltimore, USA.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Hyde
authors.affiliation: Lieber Institute for Brain Development, Baltimore, USA.
authors.forename: Joel E
authors.initials: JE
authors.lastname: Kleinman
authors.affiliation: Department of Psychiatry, University of Oxford, Oxford, United Kingdom; Oxford Health NHS Foundation Trust, Warneford Hospital, Oxford, United Kingdom.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Harrison
authors.affiliation: Department of Psychiatry, University of Oxford, Oxford, United Kingdom. Electronic address: tracy.lane@psych.ox.ac.uk.
authors.forename: Tracy A
authors.initials: TA
authors.lastname: Lane
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D001923:Brain Chemistry; D002462:Cell Membrane / Q000378:metabolism / Q000473:pathology; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D008297:Male; D018345:Mice, Knockout; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D018094:Receptors, Metabotropic Glutamate / Q000172:deficiency / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D012559:Schizophrenia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D013702:Temporal Lobe / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D055815:Young Adult
chemical_list: D000906:Antibodies; D018094:Receptors, Metabotropic Glutamate; C108822:metabotropic glutamate receptor 2; C102718:metabotropic glutamate receptor 3
keywords: Antibody validation; Group II metabotropic glutamate receptor; Human brain; Methods; Psychosis