Dynamic phospholipid signaling by G protein-coupled receptors

G protein-coupled receptors (GPCRs) control a variety of fundamental cellular processes by regulating phospholipid signaling pathways. Essential for signaling by a large number of receptors is the hydrolysis of the membrane phosphoinositide PIP₂ by phospholipase C (PLC) into the second messengers IP₃ and DAG. Many receptors also stimulate phospholipase D (PLD), leading to the generation of the versatile lipid, phosphatidic acid. Particular PLC and PLD isoforms take differential positions in receptor signaling and are additionally regulated by small GTPases of the Ras, Rho and ARF families. It is now recognized that the PLC substrate, PIP₂, has signaling capacity by itself and can, by direct interaction, affect the activity and subcellular localization of PLD and several other proteins. As expected, the synthesis of PIP₂ by phosphoinositide 5-kinases is tightly regulated as well. In this review, we present an overview of how these signaling pathways are governed by GPCRs, explain the molecular basis for the spatially and temporally organized, highly dynamic quality of phospholipid signaling, and point to the functional connection of the pathways.     

Dynamic phospholipid signaling by G protein-coupled receptors

G protein-coupled receptors (GPCRs) control a variety of fundamental cellular processes by regulating phospholipid signaling pathways. Essential for signaling by a large number of receptors is the hydrolysis of the membrane phosphoinositide PIP(2) by phospholipase C (PLC) into the second messengers IP(3) and DAG. Many receptors also stimulate phospholipase D (PLD), leading to the generation of the versatile lipid, phosphatidic acid. Particular PLC and PLD isoforms take differential positions in receptor signaling and are additionally regulated by small GTPases of the Ras, Rho and ARF families. It is now recognized that the PLC substrate, PIP(2), has signaling capacity by itself and can, by direct interaction, affect the activity and subcellular localization of PLD and several other proteins. As expected, the synthesis of PIP(2) by phosphoinositide 5-kinases is tightly regulated as well. In this review, we present an overview of how these signaling pathways are governed by GPCRs, explain the molecular basis for the spatially and temporally organized, highly dynamic quality of phospholipid signaling, and point to the functional connection of the pathways.     

Changes in G protein-coupled receptor sorting protein affinity regulate postendocytic targeting of G protein-coupled receptors

After activation, most G protein-coupled receptors (GPCRs) are regulated by a cascade of events involving desensitization and endocytosis. Internalized receptors can then be recycled to the plasma membrane, retained in an endosomal compartment, or targeted for degradation. The GPCR-associated sorting protein, GASP, has been shown to preferentially sort a number of native GPCRs to the lysosome for degradation after endocytosis. Here we show that a mutant beta(2) adrenergic receptor and a mutant mu opioid receptor that have previously been described as lacking "recycling signals" due to mutations in their C termini in fact bind to GASP and are targeted for degradation. We also show that a mutant dopamine D1 receptor, which has likewise been described as lacking a recycling signal, does not bind to GASP and is therefore not targeted for degradation. Together, these results indicate that alteration of receptors in their C termini can expose determinants with affinity for GASP binding and consequently target receptors for degradation.     

Lysophospholipid G protein-coupled receptors

The many biological responses documented for lysophospholipids that include lysophosphatidic acid and sphingosine 1-phosphate can be mechanistically attributed to signaling through specific G protein-coupled receptors. At least nine receptors have now been identified, and the total number is likely to be larger. In this brief review, we note cogent features of lysophospholipid receptors, including the current nomenclature, signaling properties, development of agonists and antagonists, and physiological functions.     

Relationship between the G protein signaling and homologous desensitization of G protein-coupled receptors

Signaling and desensitization of G protein-coupled receptor are intimately related, and measuring them separately requires certain parameters that represent desensitization independently of signaling. In this study, we tested whether desensitization requires signaling in three different receptors, beta2-adrenergic receptor (beta2AR) in S49 lymphoma cells, alpha-factor pheromone receptor (Ste2p) in Saccharomyces cerevisiae LM102 cells, and dopamine D3 receptor (D3R) in HEK-293 cells. Agonist-induced beta-arrestin translocation to the plasma membrane or receptor sequestration was measured to estimate homologous desensitization. To separate the signaling and desensitization of beta2AR, which mediates stimulation of adenylyl cyclase, S49 lymphoma cys- cells that lack the alpha subunit of Gs were used. Stimulation of beta2AR in these cells failed to increase intracellular cAMP, but beta-arrestin translocation still occurred, suggesting that feedback from beta2AR signaling is not required for homologous desensitization to occur. Agonist-induced sequestration of the yeast Ste2p-L236R, which showed reduced signaling through G protein, was not different from that of wildtype Ste2p, suggesting that the receptor signaling and sequestration are not directly linked cellular events. Both G protein coupling and D3R signaling, measured as inhibition of cAMP production, were greatly enhanced by co-expression of exogenous alpha subunit of Go (Goalpha) or adenylyl cyclase type 5 (AC5), respectively. However, agonist-induced beta-arrestin translocation, receptor phosphorylation, and sequestration were not affected by co-expression of Galphao and AC5, suggesting that the extent of signaling does not determine desensitization intensity. Taken together, our results consistently suggest that G protein signaling and homologous desensitization are independent cellular processes.     

G protein-coupled chemokine receptors induce both survival and apoptotic signaling pathways

Chemokine receptors are essential for triggering chemotaxis to immune cells; however, a number of them can also mediate death when engaged by nonchemokine ligands. When the chemokine receptor CXCR4 is engaged by stromal cell-derived factor (SDF1)alpha, it triggers cells to chemotax, and in some cell types such as neurons, causes cell death. To elucidate this dual and opposing receptor function, we have investigated whether CXCR4 activation by its chemokine SDF1alpha could lead to the simultaneous activation of both anti- and proapoptotic signaling pathways; the balance ultimately influencing cell survival. CXCR4 activation in CD4 T cells by SDF1alpha led to the activation of the prosurvival second messengers, Akt and extracellular signal-regulated protein kinase. Selective inhibition of each signal demonstrated that extracellular signal-regulated protein kinase is essential for mediating SDF1alpha-triggered chemotaxis but does not confer an antiapoptotic state. In contrast, Akt activation through CXCR4 by SDF1alpha interactions is necessary to confer resistance to apoptosis. The proapoptotic signaling pathway triggered by SDF1alpha-CXCR4 interaction involves the G(ialpha) protein-independent activation of the proapoptotic MAPK (p38). Furthermore, other chemokines and chemokine receptors also signal chemotaxis and proapoptotic effects via similar pathways. Thus, G(ialpha) protein-coupled chemokine receptors can function as death prone receptors and the balance between the above signaling pathways will ultimately mandate the fate of the activated cell.     

Protease signaling to G protein-coupled receptors: implications for inflammation and pain

Proteases, like thrombin, trypsin, cathepsins, or tryptase, can signal to cells by cleaving in a specific manner, a family of G protein-coupled receptors, the protease-activated receptors (PARs). Proteases cleave the extracellular N-terminal domain of PARs to reveal tethered ligand domains that bind to and activate the receptors. Recent evidence has supported the involvement of PARs in inflammation and pain. Activation of PAR(1), PAR(2), and PAR(4) either by proteinases or by selective agonists causes inflammation inducing most of the cardinal signs of inflammation: swelling, redness, and pain. Recent studies suggest a crucial role for the different PARs in innate immune response. The role of PARs in the activation of pain pathways appears to be dual. Subinflammatory doses of PAR(2) agonists induce hyperalgesia and allodynia, and PAR(2) activation has been implicated in the generation of inflammatory hyperalgesia. In contrast, subinflammatory doses of PAR(1) or PAR(4) increase nociceptive threshold, inhibiting inflammatory hyperalgesia, thereby acting as analgesic mediators. PARs have to be considered as an additional subclass of G protein-coupled receptors that are active participants to inflammation and pain responses and that could constitute potential novel therapeutic targets.     

Non-genomic effects of progesterone on the signaling function of G protein-coupled receptors

Progesterone at concentrations between 10 microM and 200 microM affected the calcium signaling evoked by ligand stimulation of G protein-coupled receptors expressed in several cell lines. At 160 microM progesterone the signaling of all receptors was completely abolished. The effect of progesterone was fast, reversible and was not prevented by cycloheximide indicating its non-genomic nature. Overall, the action of progesterone was more cell type-specific than receptor-specific. Our results are in contrast to a recent report [Grazzini, E., Guillon, G., Mouillac, B. and Zingg, H.H. (1998) Nature 392, 509-512] in which a direct high-affinity interaction between the oxytocin receptor and progesterone was suggested.     

Homer 2 tunes G protein-coupled receptors stimulus intensity by regulating RGS proteins and PLCbeta GAP activities

Homers are scaffolding proteins that bind G protein-coupled receptors (GPCRs), inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), ryanodine receptors, and TRP channels. However, their role in Ca2+ signaling in vivo is not known. Characterization of Ca2+ signaling in pancreatic acinar cells from Homer2-/- and Homer3-/- mice showed that Homer 3 has no discernible role in Ca2+ signaling in these cells. In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations. Thus, deletion of Homer 2 increased stimulus intensity by increasing the potency for agonists acting on various GPCRs to activate PLCbeta and evoke Ca2+ release and oscillations. This was not due to aberrant localization of IP3Rs in cellular microdomains or IP3R channel activity. Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo. Moreover, Homer 2 preferentially bound to PLCbeta in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCbeta in an in vitro reconstitution system, with minimal effect on PLCbeta-mediated PIP2 hydrolysis. These findings describe a novel, unexpected function of Homer proteins, demonstrate that RGS proteins and PLCbeta GAP activities are regulated functions, and provide a molecular mechanism for tuning signal intensity generated by GPCRs and, thus, the characteristics of [Ca2+]i oscillations.     

Olfactory receptors: G protein-coupled receptors and beyond

Sensing the chemical environment is critical for all organisms. Diverse animals from insects to mammals utilize highly organized olfactory system to detect, encode, and process chemostimuli that may carry important information critical for health, survival, social interactions and reproduction. Therefore, for animals to properly interpret and react to their environment it is imperative that the olfactory system recognizes chemical stimuli with appropriate selectivity and sensitivity. Because olfactory receptor proteins play such an essential role in the specific recognition of diverse stimuli, understanding how they interact with and transduce their cognate ligands is a high priority. In the nearly two decades since the discovery that the mammalian odorant receptor gene family constitutes the largest group of G protein-coupled receptor (GPCR) genes, much attention has been focused on the roles of GPCRs in vertebrate and invertebrate olfaction. However, is has become clear that the 'family' of olfactory receptors is highly diverse, with roles for enzymes and ligand-gated ion channels as well as GPCRs in the primary detection of olfactory stimuli.     

G protein-coupled receptors in energy homeostasis

G-protein coupled receptors (GPCRs) compromise the largest membrane protein superfamily which play vital roles in physiological and pathophysiological processes including energy homeostasis. Moreover, they also represent the up-to-date most successful drug target. The gut hormone GPCRs, such as glucagon receptor and GLP-1 receptor, have been intensively studied for their roles in metabolism and respective drugs have developed for the treatment of metabolic diseases such as type 2 diabetes (T2D). Along with the advances of biomedical research, more GPCRs have been found to play important roles in the regulation of energy homeostasis from nutrient sensing, appetite control to glucose and fatty acid metabolism with various mechanisms. The investigation of their biological functions will not only improve our understanding of how our body keeps the balance of energy intake and expenditure, but also highlight the possible drug targets for the treatment of metabolic diseases. The present review summarizes GPCRs involved in the energy control with special emphasis on their pathophysiological roles in metabolic diseases and hopefully triggers more intensive and systematic investigations in the field so that a comprehensive network control of energy homeostasis will be revealed, and better drugs will be developed in the foreseeable future.     

Mechanisms of G protein-coupled receptors regulation

Within the last two decades of studies in the ever-expanding field of GPCR signaling, challenging insights were adopted. Growing evidence now asists the shift from classical linear model of signaling towards a considerably complex network of signaling pathways with many shared proteins and cross-talks. Considering the extensive and intriguing network of pathways activated by these receptors, it is apparent that multi-level system of regulation must exist to rigorously modulate the amplitude, duration and spatial aspects of the GPCR signaling. This review summarizes the principal mechanisms of GPCR regulation and gives the overview of recent advances in this field of research.     

Structural biology of G protein-coupled receptors

More than 60% of the current drugs are based on G protein-coupled receptors. Paradoxically, high-resolution structures are not available to facilitate rational drug design. Difficulties in expression, purification, and crystallization of these transmembrane receptors are the reasons for the low success rate. Recent individual and network-based technology development has significantly improved our knowledge of structural biology and might soon bring a major breakthrough in this area.     

Toward multivalent signaling across G protein-coupled receptors from poly(amidoamine) dendrimers

Activation of the A2A receptor, a G protein-coupled receptor (GPCR), by extracellular adenosine, is antiaggregatory in platelets and anti-inflammatory. Multiple copies of an A2A agonist, the nucleoside CGS21680, were coupled covalently to PAMAM dendrimers and characterized spectroscopically. A fluorescent PAMAM-CGS21680 conjugate 5 inhibited aggregation of washed human platelets and was internalized. We envision that our multivalent dendrimer conjugates may improve overall pharmacological profiles compared to the monovalent GPCR ligands.     

The generation of nitric oxide by G protein-coupled receptors

The highly reactive free radical gas, nitric oxide, serves a variety of biomodulatory functions and has been implicated in a growing array of physiological and pathophysiological states. The striking differences between this labile substance and other, more conventional, signaling molecules highlight the tight degree of nitric oxide regulation that is required in order to maintain appropriate cellular homeostasis. The generation of nitric oxide represents a common component of the signal transduction pathways of a number of chemical signaling molecules that act via binding to G protein-coupled receptors. This review focuses on the relationship between this receptor superfamily, the generation of nitric oxide via the actions of the nitric oxide synthases and some of the inter- and intracellular roles of nitric oxide.     

Agonist-bound structures of G protein-coupled receptors

G protein-coupled receptors (GPCRs) play a major role in intercellular communication by binding small diffusible ligands (agonists) at the extracellular surface. Agonist-binding induces a conformational change in the receptor, which results in the binding and activation of heterotrimeric G proteins within the cell. Ten agonist-bound structures of non-rhodopsin GPCRs published last year defined for the first time the molecular details of receptor activated states and how inverse agonists, partial agonists and full agonists bind to produce different effects on the receptor. In addition, the structure of the β(2)-adrenoceptor coupled to a heterotrimeric G protein showed how the opening of a cleft in the cytoplasmic face of the receptor as a consequence of agonist binding results in G protein coupling and activation of the G protein.     

Splice variants of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are encoded by a vast gene superfamily, reflecting the large number of ligands that must be specifically recognized at any given cell surface. The discovery that the variety of GPCRs is further expanded through the generation of splice variants was therefore somewhat surprising. Studies of the functional consequences of alternative splicing have focused on ligand binding, signaling, constitutive activity, and downregulation. However, GPCRs also appear to interact directly with many other intracellular proteins in addition to G proteins. Intriguingly, the domains involved in these interactions are the predominant sites of variation arising through splicing.     

Desensitization of herpesvirus-encoded G protein-coupled receptors

Members of the herpesvirus family, including human cytomegalovirus (HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), encode G protein-coupled receptor (GPCR) homologs, which strongly activate classical G protein signal transduction networks within the cell. In animal models of herpesvirus infection, the viral GPCRs appear to play physiologically important roles by enabling viral replication within tropic tissues and by promoting reactivation from latency. While a number of studies have defined intracellular signaling pathways activated by herpesviral GPCRs, it remains unclear if their physiological function is subjected to the process of desensitization as observed for cellular GPCRs. G protein-coupled receptor kinases (GRK) and arrestin proteins have been recently implicated in regulating viral GPCR signaling; however, the role that these desensitization proteins play in viral GPCR function in vivo remains unknown. Here, we review what is currently known regarding viral GPCR desensitization and discuss potential biological ramifications of viral GPCR regulation by the host cell desensitization machinery.