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At the FASEB summer research conference on 'Arf Family GTPases', held in II Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.

The major G-protein coupled receptors (GPCRs) regulatory pathway involves phosphorylation of activated receptors by G-protein-coupled receptor kinases (GRKs) followed by binding of arresting proteins. The physiological functions and potential pathophysiological roles of GRKs and arrestins in human disorders are highlighted to explore the role of GRK-arrestin regulation of GPCRs in vivo.

The G protein-coupled receptor kinases (GRKs) and [beta]-arrestins, families of molecules essential to the desensitization of G protein-dependent signaling via seven-transmembrane receptors (7TMRs), have been recently shown to also transduce G protein-independent signals from receptors. However, the physiologic consequences of this G protein-independent, GRK/[beta]-arrestin-dependent signaling are largely unknown. Here, we establish that GRK/ [beta]-arrestin-mediated signal transduction via the angiotensin II (ANG) type 1A receptor (A[T.sub.1A]R) results in positive inotropic and lusitropic effects in isolated adult mouse cardiomyocytes. We used the 'biased' A[T.sub.1A]R agonist [[Sar.sup.1], [Ile.sup.4], [Ile.sup.8]-angiotensin II (SII), which is unable to stimulate [G.sub.[alpha]]q-mediated signaling, but which has previously been shown to promote [beta]-arrestin interaction with the A[T.sub.1A]R. Cardiomyocytes from WT, but not A[T.sub.1A]R-deficient knockout (KO) mice, exhibited positive inotropic and lusitropic responses to both ANG and SII. Responses of WT cardiomyocytes to ANG were dramatically reduced by protein kinase C (PKC) inhibition, whereas those to SII were unaffected. In contrast, cardiomyocytes from [beta]-arrestin2 KO and GRK6 KO mice failed to respond to SII, but displayed preserved responses to ANG. Cardiomyocytes from GRK2 heterozygous knockout mice (GRK[2.sup.+/-]) exhibited augmented responses to SII in comparison to ANG, whereas those from GRK5 KO mice did not differ from those from WT mice. These findings indicate the existence of independent [G.sub.[alpha]q/PKC- and GRK6/ [beta]-arrestin2-dependent mechanisms by which stimulation of the A[T.sub.1A]R can modulate cardiomyocyte function, and which can be differentially activated by selective receptor ligands. Such ligands may have potential as a novel class of therapeutic agents. seven-transmembrane receptors | G protein coupled receptor kinase | mice

Nitric oxide (NO) production by endothelial cell nitric oxide synthase (eNOS) in sinusoidal endothelial cells is reduced in the injured liver and leads to intrahepatic portal hypertension. We sought to understand the mechanism underlying defective eNOS function. Phosphorylation of the serine-threonine kinase Akt, which activates eNOS, was substantially reduced in sinusoidal endothelial cells from injured livers. Overexpression of Akt in vivo restored phosphorylation of Akt and production of NO and reduced portal pressure in portal hypertensive rats. We found that Akt physically interacts with G-protein-coupled receptor kinase-2 (GRK2), and that this interaction inhibits Akt activity. Furthermore, GRK2 expression increased in sinusoidal endothelial cells from portal hypertensive rats and knockdown of GRK2 restored Akt phosphorylation and NO production, and normalized portal pressure. Finally, after liver injury, GRK2-deficient mice developed less severe portal hypertension than control mice. Thus, an important mechanism underlying impaired activity of eNOS in injured sinusoidal endothelial cells is defective phosphorylation of Akt caused by overexpression of GRK2 after injury., Author(s): Songling Liu [1, 2, 4]; Richard T Premont [2]; Christopher D Kontos [2, 3]; Shoukang Zhu [2]; Don C Rockey (corresponding author) [1, 2, 4] Portal hypertension is an [...]

Dopamine supersensitivity occurs in schizophrenia and other psychoses, and after hippocampal lesions, antipsychotics, ethanol, amphetamine, phencyclidine, gene knockouts of Dbh (dopamine [beta]-hydroxylase), Drd4 receptors, Gprk6 (G protein-coupled receptor kinase 6), Comt (catechoI-O-methyltransferase), or Th-/-, [Dbh.sup.Th/+] (tyrosine hydroxylase), and in rats born by Cesarean-section. The functional state of D2, or the high-affinity state for dopamine ([D2.sup.High]), was measured in these supersensitive animal brain striata. Increased levels and higher proportions (40-900%) for [D2.sup.High] were found in all these tissues. If many types of brain impairment cause dopamine behavioral supersensitivity and a common increase in [D2.sup.High] states, it suggests that there are many pathways to psychosis, any one of which can be disrupted. addiction | dopamine receptors | gene knockouts | schizophrenia

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTP[gamma]S not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.

Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated ('desensitized') by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and [beta]-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various [beta]-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of [beta]-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3+ T and B220+ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the [beta]-arrestin2- and GRK6-deficient animals. Surprisingly, however, both T and B cells from [beta]-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that [beta]-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.

Intracellular signaling by heptahelical receptors can be brought about by mechanisms independent of G protein pathways. Some of these alternative pathways make use of receptor-interacting proteins such as arrestins and G protein-coupled receptor kinases (GRK). Arrestins and GRKs can bind to many heptahelical receptors. Arrestin and GRK-mediated formation of signaling complexes may be common to many heptahelical receptors. Other mechanisms, such as activation of GTP-binding proteins, may be relevant to a small number of heptahelical receptors but not to the majority.

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced [[Beta].sub.2]-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating [[Beta].sub.2]-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

A study was conducted to test whether palmitoylation influences the kinase activity of GRK6, a G protein-coupled receptor kinase. COS7-cells were transfected with the wild-type GRK6 DNA to determine two forms of the kinase while membranes were solubilized with Triton X-100 to obtain the palmitoylated GRK6. Results indicated that the palmitoylation of GRK6 increased the hydrophobicity and membrane association of the protein.

The [Na.sup.+]/[H.sup.+] exchanger regulatory factor (NHERF) binds to the tail of the [[Beta].sub.2]-adrenergic receptor and plays a role in adrenergic regulation of [Na.sup.+]/[H.sup.+] exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the [[Beta].sub.2] receptor. Mutagenesis studies of the [[Beta].sub.2] receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the [[Beta].sub.2] receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.

It has been possible to identify a direct agonist-promoted connection between the beta-2-adrenergic receptor and the Na+/H+ exchanger regulatory factor (NHERF), a protein that controls the activity of the Na+/H+ exchanger type 3. The binding of NHERF to the beta-2-receptor tail was found to prevent NHERF from regulating NHE3, either by making the NHERF/NHE3 interaction impossible or by changing the conformation of NHERF so that it cannot inhibit NHE3.

The beta2-adrenergic receptor mutant, Y326A, can be phosphorylated and sequestered by overexpressing G protein-coupled receptor kinase2 (GRK2) in the mutant cell. The ability of the other members of the GRK family (GRK1-6) in phosphorylating and sequestering the Y326A mutant was tested. GRK3-6 were found to aid in the phosphorylation and sequestration of the mutant cells. However, GRK1 was not able to induce phosphorylation and sequestration in Y326A mutants.

A study was done on two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases. It was shown that the cDNA encoding a hormone- and guanine nucleotide-stimulated adenylyl cyclase is stimulated by forskolin but not by betagamma subunits of guanine nucleotide-binding proteins. Another form was cloned and together with the type 5 enzyme define one distinct subfamily of mammalian adenylyl cyclases.

The progress in understanding basics of G protein-coupled receptor (GPCR) desensitization novel functional roles, patterns of brain expression and receptor specificity of G-protein coupled receptor kinases and beta arrestins in major brain functions is discussed. The results state that fine tuning of GPCR signaling would result in a more precise correction of abnormal physiological processes.

Recently, we identified a GTPase-activating protein for the ADP ribosylation factor family of small GTP-binding proteins that we call GIT1. This protein initially was identified as an interacting partner for the G protein-coupled receptor kinases, and its over-expression was found to affect signaling and internalization of the prototypical [[Beta].sub.2]-adrenergic receptor. Here, we report that GIT1 overexpression regulates internalization of numerous, but not all, G protein-coupled receptors. The specificity of the GIT1 effect is not related to the type of G protein to which a receptor couples, but rather to the endocytic route it uses. GIT1 only affects the function of G protein-coupled receptors that are internalized through the clathrin-coated pit pathway in a [Beta]-arrestin- and dynamin-sensitive manner. Furthermore, the GIT1 effect is not limited to G protein-coupled receptors because overexpression of this protein also affects internalization of the epidermal growth factor receptor. However, constitutive agonist-independent internalization is not regulated by GIT1, because transferrin uptake is not affected by GIT1 overexpression. Thus, GIT1 is a protein involved in regulating the function of signaling receptors internalized through the clathrin pathway and can be used as a diagnostic tool for defining the endocytic pathway of a receptor.