Elsevier

Biochemical Pharmacology

Volume 63, Issue 5, 1 March 2002, Pages 859-863
Biochemical Pharmacology

Association of dopamine D3 receptors with actin-binding protein 280 (ABP-280)

https://doi.org/10.1016/S0006-2952(01)00932-7Get rights and content

Abstract

Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D2 receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D3 receptors, but not with D4 receptors. Similar to the dopamine D2 receptor, the D3/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D3 receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D4 receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D3/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D3 receptor third loop, which is conserved in the D2 receptor but not in the D4 receptor. Our results demonstrate a subtype-specific regulation of dopamine D2-family receptor signaling by the cytoskeletal protein ABP-280.

Introduction

The dopamine D2 receptor regulates a variety of physiological processes including locomotion, feeding, endocrine functions, reinforcement, emotion, and cognition. Molecular cloning has identified three genes, D2, D3, and D4, that encode distinct D2-family receptors [1], [2], [3], [4], [5], [6]. However, relatively little is known about functional differences among these three D2 receptor subtypes. A number of studies have suggested that these different D2-family receptors may have preferential coupling to various G proteins [7]. Many other studies, on the other hand, have emphasized the functional significance of receptor subtype-specific protein–protein interactions. For example, the β2-adrenergic receptor has been shown to bind to Na+/H+-exchanger regulatory factor (NaH ERF) through a four amino acid motif at the receptor C-terminus. As the binding motif is present only in β2- and not in β1- or β3-adrenergic receptors, this interaction is subtype-specific [8]. Similarly, a subtype of somatostatin receptors has been shown to associate with the cortactin-binding protein [9]. Recently, proteins that bind to specific dopamine receptor subtypes have been identified. A 24 kDa single transmembrane protein, calcyon, has been shown to interact with the D1 dopamine receptor. As a result of this interaction, D1 receptors can shift effector coupling from adenylate cyclase stimulation to a robust release of intracellular calcium [10]. The γ-aminobutyric acid A (GABAA)-ligand-gated channel complex has been shown recently to selectively bind to D5 receptors, but not D1 receptors [11]. This physical association of D5 and GABAA receptors enables a mutually inhibitory functional interaction between these receptor systems.

In our previous work, we found that the D2 and the D3, but not the D4, receptor specifically binds to the cytoskeletal protein ABP-280 through its third cytoplasmic loop [12]. We have also shown that the absence of this association dramatically reduces the coupling efficiency of D2 receptor activation to the inhibition of adenylate cyclase. In the current study, we establish that the physical interaction of the D3 receptor with ABP-280 is essential for the coupling of D3 receptor activation to adenylate cyclase inhibition.

Section snippets

Yeast two-hybrid assay

A yeast two-hybrid assay was performed as described previously [12]. Briefly, the third cytoplasmic loop of the rat D3 receptor [1] was divided into four segments at residues 241, 273, and 338. Each segment was then subcloned into the GAL4 binding domain. The third cytoplasmic loop of the human D4,4 receptor [13] (residues 214–346) was also amplified by polymerase chain reaction (PCR) and subcloned in-frame into the GAL4 DNA-binding domain vector pGBT9 to generate pGBT9-D4,4. ABP-280 (residues

Results

In a previous study, we found that the C-terminal region of the D2 dopamine receptor third cytoplasmic loop is critical for binding to ABP-280 [12]. As the sequence of this region of the D2 receptor is conserved in the D3 receptor, but not in the D4 receptor, we sought to analyze whether the interaction with ABP-280 is subtype-specific. Using a yeast two-hybrid analysis, we demonstrated (Fig. 1) that the D3 receptor third cytoplasmic loop also interacted with ABP-280. However, there was no

Discussion

In this study, we have shown that dopamine D2-family receptors interact with ABP-280 in a subtype-specific manner. We found that ABP-280 binds to a region of approximately 36 amino acids in the third cytoplasmic loop, which is conserved in D2 and D3 receptors. We have further shown that the association of dopamine D2[12] and D3 receptors with ABP-280 plays an important role in coupling efficiency of both receptors with the inhibition of adenylate cyclase. In the absence of ABP-280 association, D

Acknowledgements

We thank Yu-Tian Wang for efforts in the early phase of the project. This work was supported by grants from NIH (MH57889) and the Sloan foundation (Q.-Y. Zhou).

References (20)

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