Functional and Structural Insights into the First and Second Intracellular Domains for D1-Class Dopaminergic Receptors

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Title: Functional and Structural Insights into the First and Second Intracellular Domains for D1-Class Dopaminergic Receptors
Authors: Zhang, Boyang
Date: 2017
Embargo: 2019-03-31
Abstract: Previous studies have shown that the subtype-specific pharmacological properties of D1-class receptors (D1R and D5R) can be attributed to their third intracellular domain and C-terminal tail. However, the importance of their first and second intracellular domains (IC1 and IC2) has yet to be explored. Using mutagenesis and bioinformatics, we examine the functional and structural roles of Ser/Thr spanning IC1 and IC2—most of which are conserved not only among D1-class receptors but also among other GPCRs. Mutant receptors of human D1-class receptors (hD1R and hD5R) were constructed whereby all Ser and Thr were mutated to the respective Ala and Val in the IC1 region (termed ST1 mutant receptors) and in the IC2 region (termed ST2 mutant receptors). We found that hD1-ST2 and hD5-ST2 exhibited contrasting properties of agonist affinity, constitutive activity, and dopamine potency. On the other hand, both ST2 mutants underwent internalization as wild-type but displayed weakened desensitization abilities. Homology models, which have been refined under membrane simulations, illustrate that the conserved Ser3.55 and Thr3.65 utilize their side chains to anchor the loop regions of IC2 to cytoplasmic helices. We also found multiple functional alterations in the hD1-ST1 and hD5-ST2, but in a subtype-similar manner. Mutating the conserved Thr2.39 recapitulated the ablated basal activity and drastic decrease in dopamine potency previously witnessed in the hD1-ST1. Based on the recurring theme observed in crystal structures, the side-chain of Thr2.39 may help to position IC2 to have proper contacts with the G protein. Mutating the conserved Ser2.45 was found to be solely responsible for the elevated Emax (maximal response) of the hD1-ST1. Using single point mutagenesis, we further found that breaking the potential molecular interactions of Ser2.45 in hD1R (i.e. with Asn3.42 and Trp4.50) mimicked its elevated Emax. This elevated Emax was not found to be caused by altered abilities to undergo agonist-induced desensitization or internalization relative to hD1R. Overall, our work highlights the important functional and structural roles of IC1 and IC2 that needs to be accounted for in our current canonical models of GPCR signalling. Given the conserved nature of these Ser/Thr, our work may also be pertinent towards understanding the roles of IC1 and IC2 for other GPCRs.
URL: http://hdl.handle.net/10393/35932
http://dx.doi.org/10.20381/ruor-20213
CollectionThèses - Embargo // Theses - Embargo
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