Two GPCRs (G-protein-coupled receptors), TRHR (thyrotropin-releasing hormone receptor) and β2AR (β2-adrenergic receptor), are regulated in distinct manners. Following agonist binding, TRHR undergoes rapid phosphorylation attributable to GRKs (GPCR kinases); β2AR is phosphorylated by both second messenger-activated PKA (protein kinase A) and GRKs with slower kinetics. TRHR co-internalizes with arrestin, whereas β2AR recruits arrestin, but internalizes without it. Both receptors are dephosphorylated following agonist removal, but TRHR is dephosphorylated much more rapidly while it remains at the plasma membrane. We generated chimaeras swapping the C-terminal domains of these receptors to clarify the role of different receptor regions in phosphorylation, internalization and dephosphorylation. β2AR with a TRHR cytoplasmic tail (β2AR–TRHR) and TRHR with a β2AR tail (TRHR–β2AR) signalled to G-proteins normally. β2AR–TRHR was phosphorylated well at the PKA site in the third intracellular loop, but poorly at GRK sites in the tail, whereas TRHR–β2AR was phosphorylated strongly at GRK sites in the tail (Ser355/Ser356 of the β2AR). Both chimaeric receptors exhibited prolonged, but weak, association with arrestin at the plasma membrane, but high-affinity arrestin interactions and extensive co-internalization of receptor with arrestin required a phosphorylated TRHR tail. In contrast, swapping C-terminal domains did not change the rates of phosphorylation and dephosphorylation or the dependence of TRHR dephosphorylation on the length of agonist exposure. Thus the interactions of GPCRs with GRKs and phosphatases are determined not simply by the amino acid sequences of the substrates, but by regions outside the cytoplasmic tails.

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