Selective regulation of Gq signaling by G protein-coupled receptor kinase 2: direct interaction of kinase N terminus with activated galphaq.

In this study, we investigated the regulation of different G protein-coupled receptor (GPCR)-stimulated signaling pathways by GPCR kinase 2 (GRK2). We used thyrotropin receptor, which is coupled to different G proteins, to investigate the regulation of Galphas- and Galphaq-mediated signaling (assessed by cAMP and inositol phosphate production, respectively). In transfected cells, both pathways were desensitized by GRK2. However a kinase-dead GRK2 mutant (GRK2-K220R) only decreased inositol phosphate production, indicating that GRK2 could regulate Galphaq signaling through a phosphorylation-independent mechanism. Similar results were obtained with serotonin receptor 5-hydroxytryptamine(2C), which is coupled to Galphaq. This effect was mimicked by the N-terminal domain of GRK2 (GRK2-Nter), but not by the C-terminal domain. In cells transfected with Galphaq, direct activation of Galphaq signaling (by AlF(4)(-)) was desensitized by GRK2-Nter, indicating an effect at the Galpha-level. For comparison, in parallel samples we studied a protein regulator of G protein signaling RGS4 and we found a similar regulatory profile. We therefore hypothesized that the GRK2-Nter could directly interact with the Galphaq subunit to regulate its signaling, as demonstrated for several RGS proteins. This hypothesis is further supported by the presence, within the GRK2-Nter, of an RGS homology domain. In direct binding experiments, we found that GRK2-Nter interacts with Galphaq (only when activated) but not with Galphas and Galphao. We conclude that GRK2, besides desensitizing the GPCR by phosphorylation, is able to selectively bind to Galphaq and to regulate its signaling.

Regulation of G-protein-coupled receptor kinase subtypes by calcium sensor proteins.

The process of G-protein-coupled receptor (GPCR) homologous desensitization is intrinsically related to the function of a class of S/T kinases named G-protein-coupled receptor kinases (GRK). GRK family is so far composed of six cloned members, named GRK1 to 6, which are classified into three subfamilies: GRK1 is alone in the first (rhodopsin kinase subfamily), GRK2 and 3 form the second [beta-adrenergic receptor kinase (betaARK) subfamily], and GRK4, 5, and 6 constitute the third (GRK4 subfamily). Recent studies from different laboratories have demonstrated that different calcium sensor proteins (CSP) can selectively regulate the activity of GRK subtypes. In the presence of calcium, rhodopsin kinase (GRK1) is inhibited by the photoreceptor-specific CSP recoverin through direct binding. Several other recoverin homologues (including NCS 1, VILIP 1, and hippocalcin) are also able to inhibit GRK1 in a calcium-dependent manner. The ubiquitous calcium binding protein calmodulin (CaM) can inhibit GRK5 with a high affinity (IC50=40-50 nM). A direct interaction between GRK5 and Ca2+/CaM was documented and this binding did not influence the catalytic activity of the kinase, but rather reduced GRK5 binding to the membrane. These studies suggest that CSP act as functional analogs in mediating the regulation of different GRK subtypes by Ca2+. This mechanism, however, is highly selective with respect to the GRK subtypes: GRK1, but not GRK2 and GRK5, is regulated by recoverin and other NCS, but GRK4, 5, and 6, which belong to the GRK4 subfamily are potently inhibited by CaM, which has little or no effect on members of other GRK subfamilies. Calcium-dependent inhibition of rhodopsin kinase by recoverin represents one of the mechanisms that control adaptation to light. For the other GPCR, CSP-GRK interaction provides a feedback mechanism that can modulate homologous desensitization of these receptors.

G protein-coupled receptor kinase GRK4. Molecular analysis of the four isoforms and ultrastructural localization in spermatozoa and germinal cells.

G protein-coupled receptor kinase 4 (GRK4) presents some peculiar characteristics that make it a unique member within the GRK multigene family. For example, this is the only GRK for which four splice variants (GRK4alpha, -beta, -gamma, -delta) have been identified. We developed a simple assay to study kinase activity, and we found that GRK4alpha, but not GRK4beta, -gamma, and -delta, was able to phosphorylate rhodopsin in an agonist-dependent manner. GRK4alpha kinase activity was inhibited by Ca2+/calmodulin (CaM) (IC50 = 80 nM), and a direct interaction between GRK4alpha and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. The other three GRK4 isoforms did not interact with CaM in parallel experiments. The present investigation also aimed to define cellular and ultrastructural localization of GRK4. A substantial expression of GRK4 mRNA was only found in testis and in the spermatogonia cell line GC-1 spg. Specific GRK4 immunoreactivity was only found on sperm membranes, and immunochemical and ultrastructural analyses showed that it is associated to the acrosomal membranes and to the outer mitochondrial membranes. GRK4gamma was the only detectable isoform in human sperm. We concluded that: i) only GRK4alpha can phosphorylate rhodopsin and that this activity is inhibited by CaM; ii) the other three isoforms do not phosphorylate rhodopsin and do not interact with CaM; and iii) the association of GRK4 with highly specialized sperm organelles, which are essential for fertilization, strongly indicates that this kinase is involved in this process.