D5 dopamine receptor carboxyl tail involved in D5–D2 heteromer formation

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Abstract

We have demonstrated that D5 and D2 dopamine receptors exist as heteromers in cells, and determined these receptor interact through amino acids in the cytoplasmic regions of each receptor. Specifically involved in heteromer formation we identified in the carboxyl tail of the D5 receptor three adjacent glutamic acid residues, and in intracellular loop 3 of the D2 receptor two adjacent arginine residues. Any pairing of these three D5 receptor glutamic acids were sufficient for heteromer formation. These identified residues in D5 and D2 receptors are oppositely charged and likely interact by electrostatic interactions.

Highlights

► 3 adjacent c-tail D5 receptor glutamic acids form heteromers with the D2 receptor. ► Adjacent arginines in ic3 of the D2 receptor form heteromers with the D5 receptor. ► Single amino acid changes in the D5 receptor c-tail disrupt the D5–D2 heteromers.

Introduction

Family A G protein coupled receptors (GPCRs) form heteromers [1], [2], [3]. We have reported that dopamine D1–D2 receptor heteromers exist in brain and cultured neurons [4], [5]. These heteromers were subject to conformational changes and separation by agonists [6], the heteromers reformed at the cell surface when the agonist was removed [6]. Identifying specific amino acids involved in GPCR heteromer formation has been hampered by the lack of decisive methodologies. Using our process of inserting a nuclear signal (nls) into a GPCR [7] we have identified residues involved in forming heteromers. We reported that the D1 and D2 heteromers interact by specific residues in the cytoplasmic regions. In intracellular loop 3 (ic3) of the D2 receptor, two arginine residues (274-RR) form an electrostatic interaction with vicinal glutamic residues (404-EE) in the carboxyl tail (c-tail) of the D1 receptor [8]. We also recently identified cytoplasmic residues involved in mu-delta opioid heteromers [9].

Previously we demonstrated heteromerization between the D5 and D2 receptors, our FRET analysis showed D5 and D2 receptors formed a heteromeric complex [10]. The D1 and D5 dopamine receptors share extensive overall homology (80%), however these receptors have negligible homology in their long c-tails. We questioned if D5 and D2 heteromers also form by electrostatic interactions between the D2 ic3 and D5 c-tail. In this report we have determined the specific amino acids in the cytoplasmic regions of D5 and D2 receptors involved in heteromer interactions. We demonstrated that changing the identified cytoplasmic amino acids prevented D5–D2 heteromer formation.

Section snippets

Fluorescent proteins

cDNA sequences encoding GFP, RFP were obtained from Clontech (Palo Alto, CA), and the receptor constructs generated as described [7].

Cell culture

HEK cells grown on 60 mm plates in minimum essential medium (MEM), were transfected with 0.5–2 μg cDNA using Lipofectamine (Life technologies, Rockville MD). Dopamine antagonist (+)butaclamol when used, was added to cells and cells visualized by confocal microscopy.

Microscopy

Live cells expressing GFP, and RFP fusion proteins were visualized with a LSM510 Zeiss confocal laser

Identification of the D5 dopamine receptor amino acids involved in D5–D2 heteromer formation

The D5 receptor has an extensive c-tail, extending ∼93 amino acids from the palmitoylated cysteine, Fig. 1, (consists of 26% of the total D5 receptor, the D1 receptor c-tail is 95 amino acids in length). There is negligible homology shared between the D1 and D5 receptors throughout their c-tail regions.

We incorporated an NLS into the D2 receptor (D2-nls), this did not alter the binding properties, with preserved agonist-detected high affinity and low affinity states, indicative of intact

Discussion

There are several accomplishments regarding the oligomeric structures of the D5–D2 dopamine receptors reported. (i) We determined that of three adjacent glutamic acids (429-EEE) in the c-tail of the D5 receptor, any –EE pair was sufficient to form heteromers with the D2 receptor. (ii) We determined adjacent arginines (274-RR) located in ic3 of the D2 receptor, were involved in forming heteromers with the D5 receptor. (iii) We identified single amino acid changes in the D5 receptor that

Acknowledgments

This work was partially supported by a Proof of Principle Grant from the Canadian Institutes for Health Research and National Institute on Drug Abuse Grant (DA007223). S.R.G. holds a Canada Research Chair in Molecular Neuroscience.

References (11)

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