Role of the central region of the third intracellular loop of human D1‐class dopaminergic receptors

Dopamine D1‐class receptors (D1, D5) are G‐protein coupled receptors (GPCRs) that mediate important physiological effects in the brain and periphery. The alpha‐helical amino and carboxyl ends of the third intracellular loop (IL3) of GPCRs respectively adjacent to transmembrane regions 5 (TM5) and 6 (TM6) play a pivotal role in receptor activation and coupling to G proteins. Meanwhile, it remains unclear whether the central region of IL3 linking the cytoplasmic extension of TM5 and TM6 significantly contribute to GPCR activation and G protein coupling. Here, we address this issue using deletions of either the conserved N‐terminal moiety or divergent C‐terminal moiety of the central region of IL3 of human D1‐class receptors (D1ΔN and D5ΔN; D1ΔC and D5ΔC). Agonist affinities were significantly increased and reduced at D1ΔN and D5ΔN, respectively. However, D1ΔC and D5ΔC displayed drastically increased agonist affinity. We also observed full abolition of constitutive activity of D1ΔN and D5ΔN, and a severe blunting in agonist‐induced cAMP formation. In Contrast, constitutive and agonist‐dependent activity of D1ΔC and D5ΔC were considerably increased. Overall, our study strongly suggests a critical role of the central region of IL3 in subtype‐specific activation of D1‐class dopaminergic signaling. Potentially, this may be achieved through distinct motifs located in the N‐ and C‐terminal moiety of the central region of IL3. This work is supported by a Graduate Scholarship from King Saud University (to AB) and OMHF grant (to MT).