Knockout or Knock-in? A Truncated D2 Receptor Protein Is Expressed in the Brain of Functional D2 Receptor Knockout Mice

Abstract

Null mice for the dopamine D2 receptor (D2R) have been instrumental in understanding the function of this protein. For our research, we obtained the functional D2R knockout mouse strain described initially in 1997. Surprisingly, our biochemical characterization showed that this mouse strain is not a true knockout. We determined by sequence analysis of the rapid 3′ amplification of cDNA ends that functional D2R knockout mice express transcripts that lack only the eighth exon. Furthermore, immunofluorescence assays showed a D2R-like protein in the brain of functional D2R knockout mice. We verified by immunofluorescence that the recombinant truncated D2R is expressed in HEK293T cells, showing intracellular localization, colocalizing in the Golgi apparatus and the endoplasmic reticulum, but with less presence in the Golgi apparatus compared to the native D2R. As previously reported, functional D2R knockout mice are hypoactive and insensitive to the D2R agonist quinpirole. Concordantly, microdialysis studies confirmed that functional D2R knockout mice have lower extracellular dopamine levels in the striatum than the native mice. In conclusion, functional D2R knockout mice express transcripts that lead to a truncated D2R protein lacking from the sixth transmembrane domain to the C-terminus. We share these findings to avoid future confusion and the community considers this mouse strain in D2R traffic and protein–protein interaction studies.

Immunolocalization of kappa opioid receptors in the axon initial segment of a group of embryonic mesencephalic dopamine neurons

“…Given that we could not validate primary antibodies in cell cultures obtained from knock-out since we did not have access to these KOR lines and because the D2R knock-out mice line available in our laboratory express a D2R truncated protein, recognized by our primary antibody ( Sanchez et al, 2021 ), we validated them as mentioned in Escobar et al (2017) . D2R antibody (sc-7522, Santa Cruz Biotechnology) was validated in HEK293 cells transfected with a vector encoding D2R epitope-tagged with mcherry to its c-terminus Positive signal was observed only in transfected cells, which matched mcherry fluorescence (data not shown).…”

“…Low magnification images were obtained with an appropriate 10X objective and high magnification with a 100X oil immersion objective. The primary and secondary antibodies used in mice tissue were validated in previously published studies from our laboratory ( Escobar et al, 2017 ; Sánchez et al, 2021 ).…”