Structures of Rhodopsin Kinase in Different Ligand States Reveal Key Elements Involved in G Protein-coupled Receptor Kinase Activation

Abstract

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins. Here we report six crystal structures of rhodopsin kinase (GRK1), revealing not only three distinct nucleotide-binding states of a GRK but also two key structural elements believed to be involved in the recognition of activated GPCRs. The first is the C-terminal extension of the kinase domain, which was observed in all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N terminus, observed in one of the GRK1⅐(Mg 2؉ ) 2 ⅐ATP structures. The N terminus was also clearly phosphorylated, leading to the identification of two novel phosphorylation sites by mass spectral analysis. Co-localization of the N terminus and the C-terminal extension near the hinge of the kinase domain suggests that activated GPCRs stimulate kinase activity by binding to this region to facilitate full closure of the kinase domain.

Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases

“…Crystal Structure of GRK5-Previous studies suggest that the crystallization of ligand-free GRKs is very difficult because only an ϳ8 Å crystal structure of apo-GRK1 has been reported (24). Thus, we initially focused on using nucleotides to stabilize GRK5 and facilitate crystallization.…”

“…GRK5 Is a Monomer Both in the Crystal Structure and in Solution-GRK1 and GRK6 crystallized as dimers in the asymmetric unit with the C termini of each molecule mediating a domain-swapped dimer interface (24,28,30). In contrast, GRK5 is a monomer in the crystal structure.…”

“…In contrast, the AST in GRK1 (24) and GRK6 (28) is disordered and not part of the nucleotidebinding pocket, despite the active site being occupied with ADP or ATP (AMP-PNP). AST in GRK6⅐sangivamycin is ordered, mainly due to interactions with ␣N-helix, but it is not involved in coordination of the ligand.…”

“…Several studies suggest that an N-terminal ␣-helical domain plays an important role in regulating catalytic domain closure via a process that is likely regulated by receptor binding (29 -31). Indeed, this domain has been observed in one crystal form of GRK1 (24,31) as well as in GRK6 in complex with either sangivamycin or AMP (30) and appears to stabilize catalytic domain closure. Although crystallography has provided significant insight on GRK1, -2, and -6, there have been no studies to date on the structural analysis of GRK5.…”

“…This has included defining important functional domains, including detailed mapping of interfaces such as GRK2 interaction with G␣ q (21) and G␤␥ (22) and GRK5 interaction with Ca 2ϩ /calmodulin (23). Such studies have been greatly complemented by x-ray crystallography, and structures for GRK1 (24), GRK2 (25), GRK2/G␤ 1 ␥ 2 (25,26), GRK2/G␤ 1 ␥ 2 /G␣ q (27), and GRK6 (28) have provided important insight. These structures reveal that the RH, catalytic, and C-terminal domains are in extensive contact and help to hold the kinase domain in an inactive open conformation.…”