Probing the structural determinants of the central region of third intracellular loop of human dopamine D1‐class receptor subtypes (662.9)

Abstract

Dopamine D1‐class receptors (D1, D5) are seven transmembrane receptors and play a major role in regulating important physiological effects in the brain and in the periphery. The first and the second intracellular loops are highly conserved between D1R and D5R while the third intracellular loop is the most divergent. The alpha‐helical of the amino and the carboxyl ends of the third intracellular loop (IL3) of GPCRs play a pivotal role in receptor activation and G proteins coupling. Meanwhile, it remains unclear whether the central region of IL3 linking the cytoplasmic extension of TM5 and TM6 significantly contributes to GPCR activation and G protein coupling. Here, we address this issue using either deletions of 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). Also, we test the effect of agonist‐induced desensitization on these mutants. The agonist affinities were significantly increased and decreased 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 IL3Grant Funding Source: supported by a Graduate Scholarship from King Saud University (to AB) and OMHF grant (to MT)