Functional importance of two conserved residues in intracellular loop 1 and transmembrane region 2 of Family A GPCRs: insights from ligand binding and signal transduction responses of D1 and D5 dopaminergic receptor mutants.

For many G protein-coupled receptors (GPCRs), the role of the first intracellular loop (IL1) and its connections with adjacent transmembrane (TM) regions have not been investigated. Notably, these regions harbor several polar residues such as Ser and Thr. To begin uncovering how these polar residues may contribute to the structural basis for GPCR functionality, we have designed human D1-class receptor mutants (hD1-ST1 and hD5-ST1) whereby all Ser and Thr of IL1 and IL1/TM2 juncture have been replaced by Ala and Val, respectively. Both ST1 mutants exhibited a loss of dopamine affinity but similar binding properties for inverse agonists compared to their parent receptors. As well, these mutations diminished receptor activation for both subtypes, as indicated by an ablated constitutive activity and a pronounced decrease in dopamine potency. Interestingly, both mutants exhibited enhanced dopamine-mediated maximal stimulation (Emax) of adenylyl cyclase that was at least two-fold higher than wild-type. Point mutations for hD1R revealed that the loss in dopamine affinity and potency was attributed to Thr59, while the enhanced Emax of adenylyl cyclase was directly influenced by Ser65. These two residues are conserved among many Family A GPCRs and have recurring molecular interactions among crystallized structures. As such, their functional roles for IL1 and its transition into TM2 reported herein may also be applicable to other GPCRs. Our work thus potentially highlights a structural role of Thr59 and Ser65 in the formation of critical intramolecular interactions for ligand binding and signal transduction of D1-class dopaminergic receptors.