Elsevier

Cellular Signalling

Volume 13, Issue 6, June 2001, Pages 411-416
Cellular Signalling

SH3 ligands in the dopamine D3 receptor

https://doi.org/10.1016/S0898-6568(01)00157-7Get rights and content

Abstract

It has recently been observed that G protein-coupled receptors (GPCRs) can interact with SH3 domains through polyproline motifs. These interactions appear to be involved in receptor internalization and MAPK signalling. Here we report that the third cytoplasmic loop of the dopamine D3 receptor can interact in vitro with the adaptor protein Grb2. While the amino- and carboxy-terminal SH3 domains of Grb2 separately did not interact with the D3 receptor loop, the interaction is at least partially maintained with a Grb2 mutant for the amino-terminal SH3 domain, but disrupted for a Grb2 mutant with a nonfunctional carboxy-terminal SH3 domain. The data indicate the need of structural integrity of the entire Grb2 protein for the interaction and dominant role of the carboxy-terminal SH3 domain in the interaction. Disruption of the PXXP motifs in the D3 receptor did not affect the interaction with Grb2. These results indicate that GPCRs may contain SH3 ligands that do not contain the postulated minimal consensus sequence PXXP.

Introduction

The superfamily of G protein-coupled receptors (GPCRs) mediate extracellular signals into the cell through the activation of heterotrimeric G proteins by directly promoting the GDP–GTP exchange on these signal transducers. Besides the interaction with G proteins, GPCRs have also been shown to interact with several other intracellular proteins, including arrestins, GPCR kinases (GRK), the SH2 mediated interaction of Shp2 with the angiotensin AT1 receptor and the PDZ mediated interaction of the Na+/H+ exchange regulatory factor (NHERF) with the β2-adrenergic receptor [1], [2], [3], [4], [5]. The possibility that GPCRs contain SH3 binding domains, also known as SH3 ligands, was suggested in the original discovery of SH3 ligands and their minimal sequence requirements by Ren et al. [6]. In this study [6], a putative SH3 ligand in the third cytoplasmic loop of the muscarinic M4 receptor was identified. We have recently shown that the dopamine D4 receptor can interact in vitro with the SH3–SH2 adaptor proteins Grb2 and Nck, but have also identified other receptors, like the muscarinic M4 and the β1-adrenergic receptor, that can interact with Grb2 [7].

The interaction is mediated through a large proline-rich domain in the third cytoplasmic loop of the dopamine D4 receptor with the SH3 domains of Grb2. While deletion of the D4 receptor domain that contains the SH3 ligand maintains receptor pharmacology and dopamine-activated GTPγ35S binding, this receptor mutant fails to couple functionally to the inhibition of adenylyl cyclase. This receptor is still transported to the membrane but undergoes rapid constitutive internalization. Whether the receptor interacts with Grb2 in vivo is unknown.

It has been shown that the β2-adrenergic receptor interacts in vivo with Grb2. However, this interaction appears to be mediated through a phosphotyrosine-mediated interaction which involves the Grb2 SH2 domain [8]. The interaction of the β2-adrenergic receptor with Grb2 mediates insulin-stimulated β2-adrenergic receptor internalization.

In a screening protocol for the identification of novel components of the β1-adrenergic receptor complex, it was discovered that endophilins can interact with the third cytoplasmic loop of this receptor [9]. The interaction was mediated by the C-terminal SH3 domain containing region of endophilin. Endophilin did not interact with the β2-adrenergic receptor. Overexpression of endophilin promotes agonist-mediated internalization of the β1-adrenergic receptor. Similarly, it has recently been described that the β3-adrenergic receptor can directly interact with the SH3 domain of c-Src to activate ERK in an arrestin-independent manner [10].

The dopamine D4, muscarinic M4, β1 and -3-adrenergic receptors have easy identifiable proline-rich regions in the third cytoplasmic loop that can serve as potential SH3 ligands. However, for other proteins, SH3 interactions have been discovered that are not dependent on the minimal PXXP motif. We found in our screens that the dopamine D3 receptor can also interact in vitro with Grb2. The third cytoplasmic loop of this receptor does not contain a proline-rich region and only contains two PXXP motifs that potentially can serve as SH3 ligands. Disruption of these PXXP motifs did not abolish the interaction with Grb2. These data indicate that the dopamine D3 receptor contains an SH3 ligand that does not conform to the classical PXXP consensus sequence, and suggests that the existence of SH3 ligands in GPCRs may be more widespread than predicted from the occurrence of PXXP consensus motifs.

Section snippets

Materials

Materials for plasmid isolation were purchased from Qiagen (Chatsworth, CA). Oligonucleotide primers for DNA sequencing, polymerase chain reaction (PCR) and site-directed mutagenesis were synthesized by Gibco (Burlington, ON). Restriction endonucleases were purchased from New England Biolabs (NEB, Beverly, MA). T7 RNA polymerase and RNAsin were purchased from Promega (Madison, WI). For site-directed mutagenesis, we used the Chameleon Double Stranded Site Directed Mutagenesis kit from Stratagene

In vitro protein–protein interaction assay

The protein–protein interaction has been described in detail previously [7]. In short, dopamine D3 receptor protein is synthesized in vitro in the presence of [35S]l-cysteine using a rabbit reticulocyte lysate system. GST-fusion protein expressed in Escherichia coli strains XL-1 or BL-21 was purified on glutathione beads in lysis buffer (1×PBS, 1% Triton X-100, 1% Tween 20, 10 mM DTT, 2 μg/ml aprotinin, 2 μg/ml leupeptin). For the interaction assay, a fixed amount of [35S]l-cysteine labelled

SH3 domain dopamine D3 receptor interactions

We previously reported that GPCRs can interact with SH3 domain containing proteins [7]. To detail further such interactions with the dopamine D3 receptor, we used an in vitro protein–protein interaction assay to explore the ability of several SH3 domains to interact with the entire D3 receptor (Fig. 1A). Of all the tested GST–SH3 domain fusion proteins (Nck1–255, Grb21–217, Abl65–123, Crk-II133–184, Crk-II238–290, p85α1–86, PLCγ792–851, spectrin967–1025 and c-Src84–148) only GST–Grb21–217

Discussion

Recently, it has been shown that GPCRs may interact with SH3 domains. In particular, we have explored the in vitro interaction between the SH2–SH3 adaptor protein Grb2 with the dopamine D4 receptor. This interaction was mediated by two proline-rich regions flanking a polymorphic tandem repeat region in the third cytoplasmic loop of the receptor, and appeared to play a role in receptor internalization. Furthermore, maximum strength of the interaction was dependent on the involvement of both the

Acknowledgements

We thank Dr. Mark von Zastrow for his support for some of the experimental work and critical reading of the manuscript, and Drs. J.-C. Schwartz and P. Sokoloff for their generous gift of the human dopamine D3 receptor cDNA. This work was supported by a grant of the Medical Research Council of Canada (MT-14573) (to H.H.M.V.T.) H.H.M.V.T. is a Career Scientist of the Ontario Ministry of Health.

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Both authors contributed equally to this work.

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