G Protein-Coupled Receptor Kinase 2 (GRK2) and 5 (GRK5)
Exhibit Selective Phosphorylation of the Neurotensin Receptor <i>in Vitro</i>
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Abstract
G protein-coupled
receptor kinases (GRKs) play an important role
in the desensitization of G protein-mediated signaling of G protein-coupled
receptors (GPCRs). The level of interest in mapping their phosphorylation
sites has increased because recent studies suggest that the differential
pattern of receptor phosphorylation has distinct biological consequences. <i>In vitro</i> phosphorylation experiments using well-controlled
systems are useful for deciphering the complexity of these physiological
reactions and understanding the targeted event. Here, we report on
the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1)
by GRKs under defined experimental conditions afforded by nanodisc
technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent,
whereas phosphorylation by GRK5 occurred in an activation-independent
manner. In addition, the negatively charged lipids in the immediate
vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification
of phosphorylation sites in agonist-activated NTSR1 revealed that
GRK2 and GRK5 target different residues located on the intracellular
receptor elements. GRK2 phosphorylates only the C-terminal Ser residues,
whereas GRK5 phosphorylates Ser and Thr residues located in intracellular
loop 3 and the C-terminus. Interestingly, phosphorylation assays using
a series of NTSR1 mutants show that GRK2 does not require acidic residues
upstream of the phospho-acceptors for site-specific phosphorylation,
in contrast to the β<sub>2</sub>-adrenergic and μ-opioid
receptors. Differential phosphorylation of GPCRs by GRKs is thought
to encode a particular signaling outcome, and our <i>in vitro</i> study revealed NTSR1 differential phosphorylation by GRK2 and GRK5