Insight into the Mechanism of Dopamine D1-like Receptor Activation: EVIDENCE FOR A MOLECULAR INTERPLAY BETWEEN THE THIRD EXTRACELLULAR LOOP AND THE CYTOPLASMIC TAIL

Katerina Tumova,Rafal M Iwasiow,Mario Tiberi

doi:10.1074/jbc.m208059200

Abstract

A chimeric D1A dopaminergic receptor harboring the cytoplasmic tail (CT) of the D1B subtype (D1A-CTB) has been used previously to show that CT imparts high dopamine (DA) affinity and constitutive activity to the D1B receptors. However, the D1A-CTB chimera, unlike the D1B subtype, exhibits a significantly lower DA potency for stimulating adenylyl cyclase and a drastically lower maximal binding capacity (Bmax). Here, using a functional complementation of chimeric D1-like receptors, we have identified the human D1B receptor regions regulating the intramolecular relationships that lead to an increased DA potency and contribute to Bmax. We demonstrate that the addition of variant residues of the third extracellular loop (EL3) of the human D1B receptor into D1A-CTB chimera leads to a constitutively active mutant receptor displaying an increased DA affinity, potency, and Bmax. These results strongly suggest that constitutively active D1-like receptors can adopt multiple active conformations, notably one that confers increased DA affinity with decreased DA potency and Bmax and another that imparts increased DA affinity with a strikingly increased DA potency and Bmax. Overall, we show that a novel molecular interplay between EL3 and CT regulates multiple active conformations of D1-like receptors and may have potential implications for other G protein-coupled receptor classes.

Highlights

A chimeric D1A dopaminergic receptor harboring the cytoplasmic tail (CT) of the D1B subtype (D1A-CTB) has been used previously to show that CT imparts high dopamine (DA) affinity and constitutive activity to the D1B receptors
Based upon the EL3B- and CTB-induced effects individually exerted on the D1A subtype for DA affinity, it would be expected that a chimeric D1A receptor harboring the EL3B and CTB regions would display an even higher DA affinity than that of either the wild-type D1B subtype or D1AEL3CTB chimera
We used a functional complementation of chimeric D1-like receptors to address the molecular interplay between an extracellular region, EL3, and an intracellular region, CT, in coordinating the functional properties of G protein-coupled receptor (GPCR) and potentially their multiple active conformations

Summary

Introduction

A chimeric D1A dopaminergic receptor harboring the cytoplasmic tail (CT) of the D1B subtype (D1A-CTB) has been used previously to show that CT imparts high dopamine (DA) affinity and constitutive activity to the D1B receptors. We demonstrate that the addition of variant residues of the third extracellular loop (EL3) of the human D1B receptor into D1A-CTB chimera leads to a constitutively active mutant receptor displaying an increased DA affinity, potency, and Bmax. We have shown that a structural domain (referred to as the terminal receptor locus (TRL)) encompassing the sixth and seventh transmembrane regions (TM6 and TM7), third extracellular loop (EL3) and cytoplasmic tail (CT) plays an important role in the phenotypic expression of ligand binding and G protein coupling properties of D1-like receptors [10]. Chimeric receptors harboring the CT of their respective wild-type counterparts displayed a full switch in DA affinity and constitutive activity, whereas DA potency decreased and agonist-mediated maximal activation of AC increased for both chimeras [11]. Mutagenesis studies revealed a previously unappreciated role of these receptor regions in determining the maximal binding capacity (Bmax) of D1-like subtypes (10 –13), a facet of the D1-like receptor function that may

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