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

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.

1. Introduction

Family A G protein coupled receptors (GPCRs) form heteromers [1-3]. 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.

2. Materials and methods

2.1. Fluorescent proteins

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

2.2. 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.

2.3. Microscopy

Live cells expressing GFP, and RFP fusion proteins were visualized with a LSM510 Zeiss confocal laser microscope. In each experiment 5-8 fields, containing 50-80 cells per field were evaluated and the entire experiment was repeated several times (n=3-5).

2.4. DNA Constructs

All the DNA encoding the GPCRs were human origin. Sequences encoding GPCRs were cloned into plasmids pEGFP, as described previously [7 and 11]. The D₅ carboxyl tail DNA PCR product, containing no stop codon was subcloned into vector RFP (BD Biosciences) at EcoR1 and Kpn1 and inframe with the start codon of RFP.

2.5. Receptor Constructs

The D₅ receptor constructs were prepared using the Quickchange mutagenesis kit (Stratagene) according to the manufacturer’s instructions, and as described [7 and 11]. Receptor DNA was subjected to PCR as previously reported [7]. The reaction mixture consisted of: H₂O (32 μl), 10× Pfu buffer (Stratagene) (5μl), dNTP (10mM, 5μl), DMSO (5μl), oligonucleotide primers (100ng, 1μl each), DNA template (100ng), Pfu enzyme (5U). Total volume 50μl. PCR conditions, one cycle at 94 °C for 2 min, 30-35 cycles at 94 °C for 30 sec, 55 °C for 30 sec, 72 °C for 1 min, per cycle, and then one cycle at 72 °C for 5 min. The D₂-nls and D₁-nls receptor construct was prepared as previously described [7].

3. Results

3.1. 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.

Figure 1.

Representation of the cytoplasmic intracellular tail of the D₅ dopamine receptor and the cytoplasmic intracellular third loop of the D₂ dopamine receptor. The position of the insert of 29 amino acids in the D₂ long receptor is indicated by shading.

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 receptor-G protein coupling [7].

We expressed D₅ and D₂-nls dopamine receptors in cells and demonstrated heteromer formation, Fig. 2, since the D₂-nls receptor was able to translocate the D₅ receptor to the nucleus. Despite the lack of homology in the extensive c-tails of D₁ and D₅, in the D₅ receptor c-tail there are three adjacent glutamic acids (429–EEE) in a region comparable with the glutamic acid pair (404–EE) located in the D₁ receptor, Fig. 1. The D₁ receptor glutamic acids (404-EE) were identified as forming hetromers with the D₂ receptor [8]. We wished to determine if these three glutamic acids (429-EEE) located in the c-tail of the D₅ receptor were involved in forming heteromers with the D₂ receptor.

Figure 2.

Visualization of expression of D₅ and D₂-NLS dopamine receptors.

A. D₅ (RFP) (red), expressed at the cell surface. B. D₅ (RFP) (red) and D₂-nls (GFP) (green) co-translocated to the nucleus. C. C1 (D₅)(RFP) (red) and D₂-nls (GFP) (green) did not co-translocate to the nucleus. D. C4 (D₅)(RFP) (red) and D₂-nls (GFP) (green) co-translocated to the nucleus. E. C5 (D₅)(RFP) (red) and D₂-nls (GFP) (green) co-translocated to the nucleus. F. C3 (D₅)(RFP) (red) and D₂-nls (GFP) (green) did not co-translocate to the nucleus. G. C6 (D₅)(RFP) (red) and D₂-nls (GFP) (green) co-translocated to the nucleus. H. D₅ (RFP) (red) and D₂-nls (RR to AA) (GFP) (green) did not co-translocate to the nucleus. I. D₅ (RFP) (red) and D₁-nls (GFP) (green) co-translocated to the nucleus. J. D₅ (RFP) (red) and D₁-nls (GFP) (green) co-translocated to the nucleus. Each size bar in figures showing cells indicates length of 10 μm.

We prepared a series of six substitution constructs in the D₅ receptor c-tail (Table 1A), and each construct was expressed with the D₂-nls receptor. These D5 receptors expressed alone were located predominantly in the cytoplasm. The D₅ receptor c-tail constructs C1 (429-EEE to AAA), C2 (429-EEE to EAA) and C3 (429-EEE to EAE) all failed to show D₅-D₂ heteromerization (Fig. 2 and Table 1A), since the D₂ receptor did not translocate these D₅ receptors to the nucleus. D₅-D₂ heteromer formation was observed with D₅ receptor constructs C4 (429-EEE to AEE) and C5 (429-EEE to EEA), in which each contained the vicinal –EE residues. Also in C6 construct substitution of the adjacent aspartic acid (DEEE to AEEE) did not affect D₅-D₂ heteromer formation (Fig. 2 and Table 1A). These experiments indicated a heteromer requirement of at least a pair of glutamic acids (–EE) in the D₅ receptor c-tail. Thus like D₁ receptor it appears that in the D₅ receptor the equivalently located glutamic acid pairs were also involved in heteromerization of D₅-D₂ receptors. The presence of three glutamic acids (429-EEE) in the D₅ receptor, compared to two glutamic acids in (404–EE) in the D₁ receptor, would potentially allow two positions for oligomer formation with D₂ receptor, utilizing 429-EE or 430-EE.

Table 1 A.

D5 dopamine receptor constructs.

		Heteromer
W/T	… E V D N D E E E G P F D R	Yes
C1	… E V D N D A A A G P F D R	No
C2	… E V D N D E A A G P F D R	No
C3	… E V D N D E A E G P F D R	No
C4	… E V D N D A E E G P F D R	Yes
C5	… E V D N D E E A G P F D R	Yes
C6	… E V D N A E E E G P F D R	Yes

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

We wished to determine if the arginines (274–RR) located in ic3 of the D₂ receptor were involved in forming heteromers with the D₅ receptor. These arginines, identified as being involved in D₁-D₂ heteromers, were located a distance of 59 amino acids from transmembrane 5 (TM5), Fig. 1. The D₂-nls receptor with these arginines substituted (274–RR to AA) and the D₅ receptor were co-expressed, Fig. 2. These receptors D₅ and D₂-nls (RR to AA) did not form heteromers, confirming that both D₁ and D₅ dopamine receptors utilized the same residues in the dopamine D₂ receptor ic3 for heteromer formation.

3.3 Formation of D₅-D₁ dopamine receptor heteromers

The D₁-nls and D₅ receptors were co-expressed and formed heteromers, Fig 2, since the D₅ receptor was visualized translocating with D₁-nls to the nucleus. When D₁ receptor-nls was co-expressed with D₅ receptor construct, C1 (EEE to AAA), the D₁-D₅ heteromers remained together indicating that these D₅ receptor glutamic acids residues were not involved in forming D₁-D₅ heteromers.

4. 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 disrupted the D₅-D₂ heteromers. (iv) We also determined that in the c-tail of the D₅ receptor glutamic acid residues (429-EEE) were not involved in D₅-D₁ receptor heteromer formation. Thus our GPCR-nls incorporation strategy has now enabled elucidation of cytoplasmic structural features of two dopamine receptor families D₅-D₂, and D₁-D₂ receptor heteromers [8].

Despite the overall lack of homology in the ~ninety amino acids residues in the c-tails of D₁ and D₅ receptors, contiguous glutamic acids were located in equivalent positions and each shown to be involved in forming heteromers. The D₁ and D₂ dopamine receptors form heteromers with D₅ receptor using different interacting residues. Although the residues involved in D₁-D₅ heteromers have not yet been identified, potentially involving TM regions.

Interestingly the rat D₅ receptor c-tail contains five contiguous glutamic acids, presumably involved in forming heteromers with the D₂ receptor (Table 1B). Thus heteromer formation with these various glycine pairs, (potentially forming heteromers with any of four possible –EE pairs), interact with arginines in ic3 of the D₂ dopamine receptor permitting minor variations in the conformation of the D₅-D₂ heteromer cytoplasmic structures. The mouse D₅ receptor has a total of four glutamic acids in a row then alanine and three additional contiguous glutamic acids, (Table 1B), allowing 6 different EE pairings in heteromer formation with the D₂ receptor. In comparison the D₁ dopamine receptor in human and rodent contains only a single glycine pair (404-EE).

Table 1B.

D5 dopamine receptor cytoplasmic tail sequence in mammalian species.

Human D5:	E V D N D E E E G P F D
Rat D5:	E V G E E E E E G P F D
Mouse D5:	E V G E E E E A E E E G P F D
Dog D5:	E V D K Q EE S P F D

Previously using the GPCR-nls strategy we identified discrete cytoplasmic regions in the mu and delta opioid receptors required for oligomer formation. In the carboxyl tail of the delta receptor we identified three glycine residues (-GGG), substitution of any of these residues prevented heteromer formation. In ic3 of both mu and delta receptors we identified three residues (-SVR), substitution of any of these residues prevented heteromer formation.

Thus data from our studies of four heteromer families, dopamine heteromers D₅-D₂, D₁-D₂, D₁-D₅, and mu-delta heteromers, indicate there is not a common mechanism for heteromer formation within Family A GPCRs. Receptor heteromerization arose in the cell to increase complexity, to develop and expand the utility and versatile range of functions of each individual GPCR, thus ways of interacting with other distantly related GPCRs as heteromers were opportunistically adapted. Whereas with closely related GPCRs such as D₁ or D₅, or mu and delta opioid receptors, these receptors maintained the formations used by homooligomer pairs, in forming heteromers.

In summary, we elucidated precise aspects of the cytoplasmic structure of D₅-D₂ receptor heteromers. By changing single amino acids in the D₅ receptor c-tail we succeeded is disrupting the D₅ receptor ability to form heteromers. We can now prepare D₅ and D₂ receptor expressing cells incapable of forming heteromers. Thus our work on the dopamine and opioid receptors is revealing the nature of the interactions involved in heteromers in the Family A GPCRs.

• 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