Molecular Brain Research 80 (2000) 63–74 www.elsevier.com / locate / bres Research report The splice variant D3nf reduces ligand binding to the D3 dopamine receptor: evidence for heterooligomerization a a a,c a,b,c, * Jennifer L. Elmhurst , Zhidong Xie , Brian F. O’Dowd , Susan R. George a Department of Pharmacology, Room 4358, Medical Sciences Building, University of Toronto,1 King’ s College Circle, Toronto, Ontario, Canada M5S 1A8 b Department of Medicine, University of Toronto,1 King’ s College Circle, Toronto, Ontario, Canada M5S 1A8 c Centre for Addiction and Mental Health, Toronto, Canada Accepted 23 May 2000 Abstract The D3 dopamine receptor belongs to the D2-like family of dopamine receptors. As with other members of this group, the D3 dopamine receptor gene contains introns which allow for alternative splicing of gene products. The best characterized of the human D3 dopamine receptor mRNA splice variants encodes a truncated protein called D3nf. The D3 dopamine receptor and D3nf were epitope-tagged and expressed in Sf9 insect cells by recombinant baculovirus infection. The D3 dopamine receptor showed saturable, high affinity binding of agonists and antagonists, consistent with reported D3 dopamine receptor pharmacology. When the D3 dopamine receptor and D3nf were co-expressed, the apparent density of D3 dopamine receptor expression, as determined by radioligand binding, was significantly lowered compared to D3 dopamine receptor expressed alone. This effect of D3nf was specific for the D3 dopamine receptor, since co-expression with the D2 dopamine receptor or b2-adrenoceptor had no effect on binding. Confocal immunofluorescence studies were used to confirm that both D3 dopamine receptor and D3nf were well expressed on the cell surface and densitometric analysis of cell surface membrane protein confirmed that D3nf did not significantly alter the amount of D3 dopamine receptor expressed. 125 Photoaffinity labelling with [ I]azidonemonapride showed that the amount of ligand bound by membranes co-expressing D3 dopamine receptor and D3nf was significantly less than that bound by membranes expressing D3 dopamine receptor alone. The greatest decrease in binding was observed in the D3 dopamine receptor oligomeric forms. Ligand binding to dimers and tetramers was reduced by 69 and 46%, respectively, indicating effects of a protein–protein interaction. Co-immunoprecipitation confirmed that the D3DR and D3nf interact with each other. These data indicate that D3nf heterodimerizes with the D3 dopamine receptor and decreases the capacity of D3 dopamine receptor to bind ligand.  2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Catecholamine receptors Keywords: Dopamine D3 receptor; D3nf; Splice variant; Dimers; Oligomers 1. Introduction pling to adenylyl cyclase (AC) and gene structure. The D3 dopamine receptor belongs to the D2-like subfamily and its Dopamine receptors are of interest clinically because limbic distribution in the brain implies that drugs selective they are targets in the treatment of certain neurological for this receptor may provide clinical efficacy in the disorders including schizophrenia and Parkinson’s disease. treatment of the above disorders without extrapyramidal Dopamine receptors are classified into two main groups, side effects [24]. D1-like and D2-like, based on their pharmacology, cou- As with other D2-like receptors, the D3 dopamine receptor gene contains introns which allow for the possi- bility of alternative splicing of gene products. Indeed, *Corresponding author. Tel.: 11-416-978-3367; fax: 11-416-971- several splice variants of the D3 dopamine receptor have 2868. E-mail address: s.george@utoronto.ca (S.R. George). been reported in the rat, mouse and human 0169-328X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X(00)00120-0