植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

Insect G protein-coupled receptors and signal transduction

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins (7-TM) that transduce extracellular signals into cellular physiological responses through the activation of heterotrimeric guanine nucleotide binding proteins (alpha beta gamma subunits). Their general properties are remarkably well conserved during evolution. Despite this general resemblance, a large variety of different signals are mediated via this category of receptors. Several GPCR-(sub)families have an ancient origin that is situated before the divergence of Protostomian and Deuterostomian animals. Nevertheless, an enormous diversification has occurred since then. The availability of novel sequence information is growing very rapidly as a result of molecular cloning experiments and of metazoan genome (Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens) and EST (expressed sequence tags) sequencing projects. The Drosophila Genome Sequencing Project will certainly have an important impact on insect signal transduction and receptor research. In parallel, convenient expression systems and functional assay procedures will be needed to investigate insect receptor properties and to monitor the effects of natural and artificial ligands. The study of the evolutionary aspects of G protein-coupled receptors and of their signaling pathways will probably reveal insect-specific features. More insight into these features may result in novel methods and practical applications. Arch.     

G protein-coupled receptor kinase-2 is a novel regulator of collagen synthesis in adult human cardiac fibroblasts

Cardiac fibroblasts (CF) make up 60-70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction and the transition to heart failure. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. We investigated the potential role of G protein-coupled receptor kinase-2 (GRK2) in modulating collagen synthesis by adult human CF isolated from normal and failing left ventricles. Baseline collagen synthesis was elevated in failing CF and was not inhibited by β-agonist stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in the failing CF, and expression and activity of GRK2 were increased 3-fold. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following β-agonist stimulation. In contrast, knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity in failing CF by expression of the GRK2 inhibitor, GRK2ct, or siRNA-mediated knockdown restored β-agonist-stimulated inhibition of collagen synthesis and decreased collagen synthesis in response to TGFβ stimulation. GRK2 appears to play a significant role in regulating collagen synthesis in adult human CF, and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.     

G protein-coupled receptor interacting proteins: emerging roles in localization and signal transduction

The mechanism by which G protein-coupled receptors (GPCRs) translate extracellular signals into cellular changes initially was envisioned as a simple linear model: activation of the receptor by agonist binding leads to dissociation of the heterotrimeric GTP-binding G protein into its alpha and betagamma subunits, both of which can activate or inhibit various downstream effector molecules. The plethora of recently described multidomain scaffolding proteins and accessory/chaperone molecules that interact with GPCR, including GPCR themselves as homo- or heterodimers, provides for diverse molecular mechanisms for ligand recognition, signalling specificity, and receptor trafficking. This review will summarize the recently described GPCR-interacting proteins and their individual functional roles, as understood. Implicit in the search for the functional relevance of these interactions is the expectation that enhancement or disruption of target cell-specific events could serve as highly selective therapeutic opportunities.     

Residues in the first extracellular loop of a G protein-coupled receptor play a role in signal transduction

The Saccharomyces cerevisiae pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were used as a model system to study ligand-receptor interaction. Cys-scanning mutagenesis on each residue of EL1, the first extracellular loop of Ste2p, was used to generate a library of 36 mutants with a single Cys residue substitution. Mutation of most residues of EL1 had only negligible effects on ligand affinity and biological activity of the mutant receptors. However, five mutants were identified that were either partially (L102C and T114C) or severely (N105C, S108C, and Y111C) compromised in signaling but retained binding affinities similar to those of wild-type receptor. Three-dimensional modeling, secondary structure predictions, and subsequent circular dichroism studies on a synthetic peptide with amino acid sequence corresponding to EL1 suggested the presence of a helix corresponding to EL1 residues 106 to 114 followed by two short beta-strands (residues 126 to 135). The distinctive periodicity of the five residues with a signal-deficient phenotype combined with biophysical studies suggested a functional involvement in receptor activation of a face on a 3(10) helix in this region of EL1. These studies indicate that EL1 plays an important role in the conformational switch that activates the Ste2p receptor to initiate the mating pheromone signal transduction pathway.     

Targeted overexpression of G protein-coupled receptor kinase-2 in osteoblasts promotes bone loss

To investigate the role of G protein-coupled receptor kinases (GRKs) in regulating bone formation in vivo, we overexpressed the potent G protein-coupled receptor (GPCR) regulator GRK2 in osteoblasts, using the osteocalcin gene-2 promoter to target expression to osteoblastic cells. Using the parathyroid hormone (PTH) receptor as a model system, we found that overexpression of GRK2 in osteoblasts attenuated PTH-induced cAMP generation by mouse calvaria ex vivo. This decrease in GPCR responsiveness was associated with a reduction in bone mineral density (BMD) in transgenic (TG) mice compared with non-TG littermate controls. The decrease in BMD was most prominent in trabecular-rich lumbar spine and was not observed in cortical bone of the femoral shaft. Quantitative computed tomography indicated that the loss of trabecular bone was due to a decrease in trabecular thickness, with little change in trabecular number. Histomorphometric analyses confirmed the decrease in trabecular bone volume and demonstrated reduced bone remodeling, as evidenced by a decrease in osteoblast numbers and osteoblast-mediated bone formation. Osteoclastic activity also appeared to be reduced because urinary excretion of the osteoclastic activity marker deoxypyridinoline was decreased in TG mice compared with control animals. Consistent with reduced coupling of osteoblast-mediated bone formation to osteoclastic bone resorption, mRNA levels of both osteoprotegrin and receptor activator of NF-kappaB ligand were altered in calvaria of TG mice in a pattern that would promote a low rate of bone remodeling. Taken together, these data suggest that enhancing GRK2 activity and consequently reducing GPCR activity in osteoblasts produces a low bone-turnover state that reduces bone mass.     

Real-time detection of interactions between the human oxytocin receptor and G protein-coupled receptor kinase-2

Although the oxytocin receptor (OTR) mediates many important functions including uterine contractions, milk ejection, and maternal behavior, the mechanisms controlling agonist-induced OTR desensitization have remained unclear, and attempts to demonstrate involvement of a G protein-coupled receptor kinase (GRK) have so far failed. Using the OTR as a model, we demonstrate here directly for the first time the dynamics of agonist-induced interactions of a GRK with a G protein-coupled receptor in real time, using time-resolved bioluminescence resonance energy transfer. GRK2/receptor interactions started within 4 sec, peaked at 10 sec, and decreased to less than 40% within 8 min. By contrast, beta-arrestin/OTR interactions initiated only at 10 sec, reached plateau levels at 120 sec, but remained stable with little decrease thereafter. Physical GRK2/OTR association was further demonstrated by coimmunoprecipitation of endogenous GRK2 with activated OTR. In COS-7 cells, which express low levels of GRK2 and beta-arrestin, overexpression of GRK2 and beta-arrestin increased receptor phosphorylation, desensitization, and internalization to the high levels observed in human embryonic kidney 293 cells. By contrast, specific inhibition of endogenous GRK2 by dominant-negative mutants robustly inhibited OTR phosphorylation and internalization as well as arrestin/OTR interactions. These data characterize the temporal and causal relationship of GRK-2/OTR and beta-arrestin/OTR interactions and establish GRK/OTR interaction as a prerequisite for beta-arrestin-mediated OTR desensitization.     

The ubiquitin-like protein PLIC-2 is a negative regulator of G protein-coupled receptor endocytosis

The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and β-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics.     

Sphingosylphosphorylcholine and lysophosphatidylcholine: G protein-coupled receptors and receptor-mediated signal transduction

In recent years, certain lysophospholipids (lyso-PLs) have been recognized as important cell signaling molecules. Among them, two phosphorylcholine-containing lyso-PLs, sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), have been shown to be involved in many cellular processes and are produced under physiological and pathological conditions. Although signaling properties of SPC and LPC have been studied in a variety of cellular systems, specific cell membrane receptors for SPC and LPC have not been identified previously. Recently, ovarian cancer G protein-coupled receptor 1 (OGR1, also known as GPR68), G protein-coupled receptor 4 (GPR4), and G2A have been identified as receptors for SPC and LPC. The signaling and ligand-binding properties of these receptors are reviewed here. These discoveries provide an intriguing opportunity and a novel approach in studying the pathophysiological roles of SPC and LPC and their receptors.     

Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 （GRK2） is an important serine/threonine-kinase regulating different membrane receptors and intraceUular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts, overexpression of a GRK2 kinase-deficient mutant （K220R） caused precocious differentiation of ceUs into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2-overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.     

The platelet-derived growth factor receptor-beta phosphorylates and activates G protein-coupled receptor kinase-2. A mechanism for feedback inhibition

G protein-coupled receptor kinase-2 (GRK2) serine-phosphorylates the platelet-derived growth factor receptor-beta (PDGFRbeta), and thereby diminishes signaling by the receptor. Because activation of GRK2 may involve phosphorylation of its N-terminal tyrosines by c-Src, we tested whether the PDGFRbeta itself could tyrosine-phosphorylate and activate GRK2. To do so, we used wild type (WT) and Y857F mutant PDGFRbetas in HEK cells, which lack endogenous PDGFRs. The Y857F PDGFRbeta autophosphorylates normally but does not phosphorylate exogenous substrates. Although PDGF-stimulated Y857F and WT PDGFRbetas activated c-Src equivalently, the WT PDGFRbeta tyrosine-phosphorylated GKR2 60-fold more than the Y857F PDGFRbeta in intact cells. With purified GRK2 and either WT or Y857F PDGFRbetas immunoprecipitated from HEK cells, GRK2 tyrosyl phosphorylation was PDGF-dependent and required the WT PDGFRbeta, even though the WT and Y857F PDGFRbetas autophosphorylated equivalently. This PDGFRbeta-mediated GRK2 tyrosyl phosphorylation enhanced GRK2 activity: GRK2-mediated seryl phosphorylation of the PDGFRbeta was 9-fold greater for the WT than for the Y857F in response to PDGF, but equivalent when GRK2 was activated by sequential stimulation of beta2-adrenergic and PDGF-beta receptors. Furthermore, both PDGFRbeta-mediated GRK2 tyrosyl phosphorylation and GRK2-mediated PDGFRbeta seryl phosphorylation were reduced approximately 50% in intact cells by mutation to phenylalanine of three tyrosines in the N-terminal domain of GRK2. We conclude that the activated PDGFRbeta itself phosphorylates GRK2 tyrosyl residues and thereby activates GRK2, which then serine-phosphorylates and desensitizes the PDGFRbeta.     

G protein-coupled receptor oligomerization provides the framework for signal discrimination

The idea that G protein-coupled receptors (GPCRs) may undergo homo- or hetero-oligomerization, although highly controversial up to a few years ago, has recently gained wide acceptance. The recognition that GPCRs may exhibit either dimeric or oligomeric structures is based upon a large body of biochemical and biophysical evidence. While much effort has been spent to demonstrate the mechanism(s) by which GPCRs interact with each other, the physiological relevance of this phenomenon remains rather elusive. GPCR oligomerization has been proposed to play a role in receptor ontogeny by either chaperoning protein folding or controlling trafficking to the cell surface. However, the acquisition of these roles does not rule out the possibility that oligomeric receptors may have additional functions, once they are brought to the cell surface. Herein, we propose that protein-protein as well as protein-lipid interactions may provide the structural basis for organizing distinct cell compartments along the plasma membrane where different extracellular signals may be perceived and discriminated.     

G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton

G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes activated G protein-coupled receptors (GPCRs). Here, we identify ezrin as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates glutathione S-transferase (GST)-ezrin, but not an ezrin fusion protein lacking threonine 567 (T567), in vitro. These results suggest that T567, the regulatory phosphorylation site responsible for maintaining ezrin in its active conformation, represents the principle site of GRK2-mediated phosphorylation. Two lines of evidence indicate that GRK2-mediated ezrin-radixinmoesin (ERM) phosphorylation serves to link GPCR activation to cytoskeletal reorganization. First, in Hep2 cells muscarinic M1 receptor (M1MR) activation causes membrane ruffling. This ruffling response is ERM dependent and is accompanied by ERM phosphorylation. Inhibition of GRK2, but not rho kinase or protein kinase C, prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalization of the beta2-adrenergic receptor (beta2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized beta2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor binding to the beta2AR. Inhibition of ezrin function impedes beta2AR internalization, further linking GPCR activation, GRK activity, and ezrin function. Overall, our results suggest that GRK2 serves not only to attenuate but also to transduce GPCR-mediated signals.     

Epstein-Barr virus-encoded BILF1 is a constitutively active G protein-coupled receptor

Both beta- and gammaherpesviruses encode G protein-coupled receptors (GPCRs) with unique pharmacological phenotypes and important biological functions. An example is ORF74, the gamma2-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded GPCR, which is highly constitutively active and considered the key oncogene in Kaposi's sarcoma pathogenesis. In contrast, the current annotation of the Epstein-Barr virus (EBV) genome does not reveal any GPCR homolog encoded by this human oncogenic gamma1-herpesvirus. However, by employing bioinformatics, we recognized that the previously established EBV open reading frame BILF1 indeed encodes a GPCR. Additionally, BILF1 is a member of a new family of related GPCRs exclusively encoded by gamma1-herpesviruses. Expression of hemagglutinin-tagged BILF1 in the HEK293 epithelial cell line revealed that BILF1 is expressed as an approximately 50-kDa glycosylated protein. Immunocytochemistry and confocal microscopy revealed that BILF1 localizes predominantly to the plasma membrane, similar to the localization of KSHV ORF74. Using chimeric G proteins, we found that human and rhesus EBV-encoded BILF1 are highly potent constitutively active receptors, activating Galphai. Furthermore, BILF1 is able to inhibit forskolin-triggered CREB activation via stimulation of endogenous G proteins in a pertussis toxin-sensitive manner, verifying that BILF1 signals constitutively through Galphai. We suggest that EBV may use BILF1 to regulate Galphai-activated pathways during viral lytic replication, thereby affecting disease progression.     

The orphan G protein-coupled receptor 161 is required for left-right patterning

Gpr161 (also known as RE2) is an orphan G protein-coupled receptor (GPCR) that is expressed during embryonic development in zebrafish. Determining its biological function has proven difficult due to lack of knowledge regarding its natural or synthetic ligands. Here, we show that targeted knockdown of gpr161 disrupts asymmetric gene expression in the lateral plate mesoderm, resulting in aberrant looping of the heart tube. This is associated with elevated Ca²⁺ levels in cells lining the Kupffer's vesicle and normalization of Ca²⁺ levels, by over-expression of ncx1 or pmca-RNA, is able to partially rescue the cardiac looping defect in gpr161 knockdown embryos. Taken together, these data support a model in which gpr161 plays an essential role in left-right (L-R) patterning by modulating Ca²⁺ levels in the cells surrounding the Kupffer's vesicle.     

Interaction of neurochondrin with the melanin-concentrating hormone receptor 1 interferes with G protein-coupled signal transduction but not agonist-mediated internalization

Screening of a human brain cDNA library using the C-terminal tail of the melanin-concentrating hormone receptor 1 (MCHR1) as bait in a yeast two-hybrid assay resulted in the identification of the neurite-outgrowth related factor, neurochondrin. This interaction was verified in overlay, pulldown, and co-immunoprecipitation assays. Deletion mapping confined the binding to the C terminus of neurochondrin and to the proximal C terminus of MCHR1, a region known to be involved in G protein binding and signal transduction. This region of the MCHR1 is also able to interact with the actin- and intermediate filament-binding protein, periplakin. Interactions of MCHR1 with neurochondrin and periplakin were competitive, indicating that these two proteins bind to overlapping regions of MCHR1. Although neurochondrin did not interfere with melanin-concentrating hormone-mediated internalization of the receptor, it did inhibit G protein-coupled signal transduction via both Galpha(i/o) and Galpha(q/11) family G proteins as measured by each of melanin-concentrating hormone-induced G protein-activated inwardly rectifying K(+) channel activity of voltage-clamped amphibian oocytes, by calcium mobilization in transfected mammalian cells, and by reduction in the capacity of melanin-concentrating hormone to promote binding of [(35)S]guanosine 5'-3-O-(thio)triphosphate to both Galpha(o1) and Galpha(11). Immunohistochemistry revealed co-expression of neurochondrin and MCHR1 within the rodent brain, suggesting that neurochondrin may be involved in the regulation of MCHR1 signaling and play a role in modulating melanin-concentrating hormone-mediated functions in vivo.     

MAPK-dependent degradation of G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.     

Cardiomyocyte-restricted inhibition of G protein-coupled receptor kinase-3 attenuates cardiac dysfunction after chronic pressure overload

Transgenic mice with cardiac-specific expression of a peptide inhibitor of G protein-coupled receptor kinase (GRK)3 [transgenic COOH-terminal GRK3 (GRK3ct) mice] display myocardial hypercontractility without hypertrophy and enhanced α(1)-adrenergic receptor signaling. A role for GRK3 in the pathogenesis of heart failure (HF) has not been investigated, but inhibition of its isozyme, GRK2, has been beneficial in several HF models. Here, we tested whether inhibition of GRK3 modulated evolving cardiac hypertrophy and dysfunction after pressure overload. Weight-matched male GRK3ct transgenic and nontransgenic littermate control (NLC) mice subjected to chronic pressure overload by abdominal aortic banding (AB) were compared with sham-operated (SH) mice. At 6 wk after AB, a significant increase of cardiac mass consistent with induction of hypertrophy was found, but no differences between GRK3ct-AB and NLC-AB mice were discerned. Simultaneous left ventricular (LV) pressure-volume analysis of electrically paced, ex vivo perfused working hearts revealed substantially reduced systolic and diastolic function in NLC-AB mice (n = 7), which was completely preserved in GRK3ct-AB mice (n = 7). An additional cohort was subjected to in vivo cardiac catheterization and LV pressure-volume analysis at 12 wk after AB. NLC-AB mice (n = 11) displayed elevated end-diastolic pressure (8.5 ± 3.1 vs. 2.9 ± 1.2 mmHg, P < 0.05), reduced cardiac output (3,448 ± 323 vs. 4,488 ± 342 μl/min, P < 0.05), and reduced dP/dt(max) and dP/dt(min) (both P < 0.05) compared with GRK3ct-AB mice (n = 16), corroborating the preserved cardiac structure and function observed in GRK3ct-AB hearts assessed ex vivo. Increased cardiac mass and myocardial mRNA expression of β-myosin heavy chain confirmed the similar induction of cardiac hypertrophy in both AB groups, but only NLC-AB hearts displayed significantly elevated mRNA levels of brain natriuretic peptide and myocardial collagen contents as well as reduced β(1)-adrenergic receptor responsiveness to isoproterenol, indicating increased LV wall stress and the transition to HF. Inhibition of cardiac GRK3 in mice does not alter the hypertrophic response but attenuates cardiac dysfunction and HF after chronic pressure overload.