Two amino acids in each of D1 and D2 dopamine receptor cytoplasmic regions are involved in D1–D2 heteromer formation
Highlights
► Pair of adjacent arginines of the D2 receptor involved in forming heteromers with the D1 receptor. ► D2 receptor with one arginine substituted did not form heteromers with the D1 receptor. ► Pair of adjacent glutamic acids in the D1 carboxyl tail involved in forming D1–D2 heteromers. ► D1 receptor with one of the glutamic acids substituted did not form D1–D2 heteromers. ► Aspartic acid substituted for glutamic acid in the D1 carboxyl tail did not form heteromers.
Introduction
Family A G protein coupled receptors (GPCRs) form heteromers [1], [2], [3]. We reported that D1–D2 receptor heteromers exist in brain and cultured neurons [4], [5]. We showed receptor activation within D1–D2 heteromers generated a Gq-mediated calcium signal [4], [6], [7]. We have determined that D1–D2 heteromers were subject to conformational changes and separation by dopamine or receptor-selective agonists [8]. We also reported that the D1 and D2 receptor heteromers reform at the cell surface when the agonist was removed [8]. These data provided evidence of the fate of a heteromer following agonist activation and demonstrated a unique regulation of GPCRs at the cell surface. However, many fine structural details of how D1–D2 heteromers dynamically interact remain unknown. In this report we have determined the precise amino acid interactions maintaining D1 and D2 receptors in a D1–D2 receptor complex. Our ultimate goal is the understanding of the physiological relevance of GPCR:GPCR heteromers, one of the leading questions in the GPCR field.
Progress in the fundamental area of GPCR oligomer structural investigation has been hampered by the lack of decisive methods for determining the interacting heteromer interface. We overcame technical challenges by the following process: a nuclear localization sequence (NLS) was inserted into the D2 receptor. Strategic placement of the NLS rendered this D2-NLS receptor conformationally sensitive, so that interacting ligands retained the receptor at the cell surface [9]. D2-NLS and the D1 receptors were coexpressed and following ligand removal, the D2-NLS receptor translocated with the D1 receptor from the cell surface. We demonstrated that as the D2-NLS receptor translocated with the D1 receptor this provided a tool to study receptor:receptor dynamic interactions in a cell [9]. By this strategy we sought to reveal the structural basis for the D1–D2 receptor interaction. By co-expressing D2-NLS and D1 receptors the contributions of various cytoplasmic regions of these receptors to heteromer formation was investigated.
In this report, we have determined the precise amino acids in the cytoplasmic regions of both the D1 and D2 receptors involved in their heteromeric interactions. Activation of the heteromer contributes to conformational changes in the receptors within the oligomer. We have now identified these residues affected by agonist induced conformational changes. Also we identified that changing a single amino acid in the intracellular loop 3 of the D2 receptor or in the carboxyl tail of the D1 receptor prevented D1–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 [9]. The YFP vector was obtained from BD Biosciences.
Cell culture
HEK cells grown to confluence on 60 mm plates in minimum essential medium (MEM), and were transfected with 0.5–2 μg cDNA using Lipofectamine (Life Technologies, Rockville MD).
Microscopy
Live cells expressing GFP, RFP and YFP fusion proteins were visualized with a LSM510 Zeiss confocal laser microscope. In each experiment 5–8
Binding and expression properties of the D2-NLS receptor and D1-NLS receptors
The incorporation of NLS into the D2 receptor did not alter the binding properties, with preserved agonist-detected high affinity and low affinity states, indicative of intact receptor-G protein coupling. The D2 receptor had a KHigh value of 1.51 × 10−9 M and KLow of 6.67 × 10−6 M for quinpirole. Similarly the D2-NLS receptor had a KHigh value of 3.22 × 10−9 M and KLow of 4.16 × 10−6 M for quinpirole [9].
The incorporation of the NLS into the D1 receptor did not alter the binding pocket of the receptor,
Discussion
There are several significant and unique accomplishments regarding the oligomeric structures of the D1–D2 dopamine receptors reported here. (i) We determined that a pair of adjacent arginines of the D2 receptor, located in the third cytoplasmic loop, were involved in forming heteromers with the D1 receptor. (ii) We determined that both arginines were required, a D2 receptor with one of the arginines substituted did not form heteromers with the D1 receptor. (iii) We determined that the
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. The authors thank Hong Fan Qian for preparation of Fig. 1.
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