Abstract
G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5'-adenylyl β,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.
Highlights
GRK4 mediates G-protein-coupled receptor (GPCR) phosphorylation and is implicated in hypertension
The structure of GRK4␣ is similar to other G-protein-coupled receptor kinases (GRK), slight differences exist within the regulator of G-protein signaling (RGS) homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail
We present the structure of a hypertension-associated A486V variant of GRK4␣ bound to an ATP analog
Summary
GRK4 mediates GPCR phosphorylation and is implicated in hypertension. Results: The crystal structure of human GRK4␣ A486V is presented. The role of GRK4 in hypertension may not be confined to dopamine receptor signaling, as GRK4 single nucleotide polymorphisms are associated with increased expression of the angiotensin II type 1 receptor, which is another important determinant of blood pressure [8, 13]. Structures of each GRK would aid in the structure-guided design of selective GRK modulators for multiple disease indications To address this gap, we present the 2.6 Å resolution structure of the hypertension-associated A486V polymorphic variant of GRK4␣ crystallized in the presence of the nonhydrolyzable ATP analog AMPPNP. We present enhanced autophosphorylation by A486V as a possible mechanism for increased activity of the hypertension-related A486V mutation These data unmask unique features of GRK4 within the GRK family and provide insights into the origins of substrate specificity and promiscuity in this important family of kinases
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