Abstract
To delineate the structural determinants involved in the constitutive activation of the D1 receptor subtypes, we have constructed chimeras between the D1A and D1B receptors. These chimeras harbored a cognate domain corresponding to transmembrane regions 6 and 7 as well as the third extracellular loop (EL3) and cytoplasmic tail, a domain referred herein to as the terminal receptor locus (TRL). A chimeric D1A receptor harboring the D1B-TRL (chimera 1) displays an increased affinity for dopamine that is indistinguishable from the wild-type D1B receptor. Likewise, a chimeric D1B receptor containing the D1A-TRL cassette (chimera 2) binds dopamine with a reduced affinity that is highly reminiscent of the dopamine affinity for the wild-type D1A receptor. Furthermore, we show that the agonist independent activity of chimera 1 is identical to the wild-type D1B receptor whereas the chimera 2 displays a low agonist independent activity that is indistinguishable from the wild-type D1A receptor. Dopamine potencies for the wild-type D1A and D1B receptor were recapitulated in cells expressing the chimera 2 or chimera 1, respectively. However, the differences observed in agonist-mediated maximal activation of adenylyl cyclase elicited by the D1A and D1B receptors remain unchanged in cells expressing the chimeric receptors. To gain further mechanistic insights into the structural determinants of the TRL involved in the activation properties of the D1 receptor subtypes, we have engineered two additional chimeric D1 receptors that contain the EL3 region of their respective cognate wild-type counterparts (hD1A-EL3B and hD1B-EL3A). In marked contrast to chimera 1 and 2, dopamine affinity and constitutive activation were partially modulated by the exchange of the EL3. Meanwhile, hD1A-EL3B and hD1B-EL3A mutant receptors display a full switch in the agonist-mediated maximal activation, which is reminiscent of their cognate wild-type counterparts. Overall, our studies suggest a fundamental role for the TRL in shaping the intramolecular interactions implicated in the constitutive activation and coupling properties of the dopamine D1 receptor subtypes.
Highlights
The classical paradigm for G protein-coupled receptor (GPCR)1 activation is described by the binding of an agonist to an inactive receptor state (R)
We have described that replacement of a variant amino acid found in the carboxyl end of the third cytoplasmic loop of the D1A by the one found in the D1B receptor can induce partially the constitutive activation of the D1A receptor [10]
This paper is available on line at http://www.jbc.org domain regulating the activation process of the D1A and D1B receptor but demonstrates that the molecular determinants involved in the GPCR activation properties can be separated
Summary
The classical paradigm for G protein-coupled receptor (GPCR) activation is described by the binding of an agonist to an inactive receptor state (R). A mutant D1B receptor harboring the variant D1A amino acid exhibits a decreased level of constitutive activity as well as the binding and coupling properties similar to those of the wild-type D1A receptor [10]. This paper is available on line at http://www.jbc.org domain regulating the activation process of the D1A and D1B receptor but demonstrates that the molecular determinants involved in the GPCR activation properties (constitutive activation, agonist potency, and intrinsic efficacy) can be separated.
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