Methylation-related metabolic effects of D4 dopamine receptor expression and activation

Verna-Ann Power-Charnitsky,Malav S Trivedi,Richard C Deth,Mostafa I Waly,Nathaniel W Hodgson

doi:10.1038/s41398-019-0630-3

Abstract

D4 dopamine receptor (D4R) activation uniquely promotes methylation of plasma membrane phospholipids, utilizing folate-derived methyl groups provided by methionine synthase (MS). We evaluated the impact of D4R expression on folate-dependent phospholipid methylation (PLM) and MS activity, as well as cellular redox and methylation status, in transfected CHO cells expressing human D4R variants containing 2, 4, or 7 exon III repeats (D4.2R, D4.4R, D4.7R). Dopamine had no effect in non-transfected CHO cells, but increased PLM to a similar extent for both D4.2R- and D4.4R-expressing cells, while the maximal increase was for D4.7R was significantly lower. D4R expression in CHO cells decreased basal MS activity for all receptor subtypes and conferred dopamine-sensitive MS activity, which was greater with a higher number of repeats. Consistent with decreased MS activity, D4R expression decreased basal levels of methylation cycle intermediates methionine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH), as well as cysteine and glutathione (GSH). Conversely, dopamine stimulation increased GSH, SAM, and the SAM/SAH ratio, which was associated with a more than 2-fold increase in global DNA methylation. Our findings illustrate a profound influence of D4R expression and activation on MS activity, coupled with the ability of dopamine to modulate cellular redox and methylation status. These previously unrecognized signaling activities of the D4R provide a unique link between neurotransmission and metabolism.

Highlights

Previous studies demonstrated the unique ability of D4 dopamine receptors (D4Rs) to carry out phospholipid methylation (PLM) in response to dopamine stimulation.[1,2,3,4] PLM activity involves conformation-dependent methyl donation by a methionine side chain in the cytoplasmic extension of transmembrane helix #6 (M313 in the D4.4R), in a cyclic process analogous to the welldescribed methionine-based methylation cycle (Fig. 1a)
We examined the impact of D4R expression and dopamine exposure on D4R-mediated PLM and methionine synthase (MS) activity, as well as redox and methylation status, using stably transfected CHO cells expressing D4.2R, D4.4R, or D4.7R
Dopamine stimulation of mitogen-activate protein (MAP) kinase phosphorylation, mediated by G protein signaling, was similar for D4.2R, D4.4R, and D4.7R, further indicating that PLM is a signaling response that differentiates the D4.7R from D4.2R and D4.4 subtypes

Summary

Introduction

Previous studies demonstrated the unique ability of D4 dopamine receptors (D4Rs) to carry out phospholipid methylation (PLM) in response to dopamine stimulation.[1,2,3,4] PLM activity involves conformation-dependent methyl donation by a methionine side chain in the cytoplasmic extension of transmembrane helix #6 (M313 in the D4.4R), in a cyclic process analogous to the welldescribed methionine-based methylation cycle (Fig. 1a). The D4R-mediated PLM cycle depends on replenishing methyl groups from 5-methyltetrahydrofolate (methylTHF) provided by methionine synthase (MS), and requires methionine adenosyltransferase (MAT). S-adenosylhomocysteine hydrolase (SAHH).[1] MS transfers a methyl group from methylTHF to restore the D4R methionine residue (D4MET), in addition to its canonical role in converting free homocysteine (HCY) to methionine. These two competing reactions imply that D4R-mediated dopaminestimulated PLM activity could influence canonical Sadenosylmethionine (SAM)-dependent methionine cycle activity. When MS activity decreases during oxidative stress, a greater proportion of HCY is converted to cysteine via transsulfuration, augmenting synthesis of the antioxidant glutathione (GSH) (Fig. 1a). Decreased MS activity leads to depletion of SAM, the methyl donor for well over 200 different methylation reactions, including D4R-independent PLM and DNA methylation

Methods

Results

Conclusion