3 research outputs found
G Protein-Coupled Receptor Kinase 2 (GRK2) and 5 (GRK5) Exhibit Selective Phosphorylation of the Neurotensin Receptor <i>in Vitro</i>
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
Structure-Based Design of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors Based on Paroxetine
In heart failure, the β-adrenergic
receptors (βARs)
become desensitized and uncoupled from heterotrimeric G proteins.
This process is initiated by G protein-coupled receptor kinases (GRKs),
some of which are upregulated in the failing heart, making them desirable
therapeutic targets. The selective serotonin reuptake inhibitor, paroxetine,
was previously identified as a GRK2 inhibitor. Utilizing a structure-based
drug design approach, we modified paroxetine to generate a small compound
library. Included in this series is a highly potent and selective
GRK2 inhibitor, <b>14as</b>, with an IC<sub>50</sub> of 30 nM
against GRK2 and greater than 230-fold selectivity over other GRKs
and kinases. Furthermore, <b>14as</b> showed a 100-fold improvement
in cardiomyocyte contractility assays over paroxetine and a plasma
concentration higher than its IC<sub>50</sub> for over 7 h. Three
of these inhibitors, including <b>14as</b>, were additionally
crystallized in complex with GRK2 to give insights into the structural
determinants of potency and selectivity of these inhibitors
Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility
G protein-coupled receptor kinase 2 (GRK2) is a well-established
therapeutic target for the treatment of heart failure. Herein we identify
the selective serotonin reuptake inhibitor (SSRI) paroxetine as a
selective inhibitor of GRK2 activity both <i>in vitro</i> and in living cells. In the crystal structure of the GRK2·paroxetine–Gβγ
complex, paroxetine binds in the active site of GRK2 and stabilizes
the kinase domain in a novel conformation in which a unique regulatory
loop forms part of the ligand binding site. Isolated cardiomyocytes
show increased isoproterenol-induced shortening and contraction amplitude
in the presence of paroxetine, and pretreatment of mice with paroxetine
before isoproterenol significantly increases left ventricular inotropic
reserve <i>in vivo</i> with no significant effect on heart
rate. Neither is observed in the presence of the SSRI fluoxetine.
Our structural and functional results validate a widely available
drug as a selective chemical probe for GRK2 and represent a starting
point for the rational design of more potent and specific GRK2 inhibitors