David R. Manning

Differential Coupling of the Sphingosine 1-Phosphate Receptors Edg-1, Edg-3, and H218/Edg-5 to the Gi, Gq, and G12 Families of Heterotrimeric G Proteins

Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [35S]GTPγS binding assay to investigate whether the variety of actions exerted through Edg-1, a recently identified receptor for S1P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-1 activates only members of the Gi family, and not Gs, Gq, G12, or G13. We additionally established that Edg-1 activates Gi in response not only to S1P but also sphingosylphosphorylcholine; no effects of lysophosphatidic acid through Edg-1 were evident. Our assays further revealed a receptor(s) for S1P endogenous to HEK293 cells that mediates activation of G13 as well as Gi. Because several of the biological actions of S1P are assumed to proceed through the G12/13 family, we tested whether Edg-3 and H218/Edg-5, two other receptors for S1P, might have a broader coupling profile than Edg-1. Indeed, Edg-3 and H218/Edg-5 communicate not only with Gi but also with Gq and G13. These studies represent the first characterization of S1P receptor activity through G proteins directly and establish fundamental differences in coupling.

Regulation of G proteins by covalent modification

Heterotrimeric G protein α,β, and γ subunits are subject to several kinds of co- and post-translational covalent modifications. Among those relevant to G protein-coupled receptor signaling in normal cell function are lipid modifications and phosphorylation. N-myristoylation is a co-translational modification occurring for members of the Gi family of Gα subunits, while palmitoylation is a post-translational modification that occurs for these and most other Gα subunits. One or both modifications are required for plasma membrane targeting and contribute to regulating strength of interaction with the Gβγ heterodimer, effectors, and regulators of G protein signaling (RGS proteins). Gα subunits, including those with transforming activity, are often inactive when unable to be modified with lipids. The reversible nature of palmitoylation is intriguing in this regard, as it lends itself to a regulation integrated with the activation state of the G protein. Several Gα subunits are substrates for phosphorylation by protein kinase C and at least one is a substrate for phosphorylation by the p21-activated protein kinase. Phosphorylation in both instances inhibits the interactions of these subunits with the Gβγ heterodimer and RGS proteins. Several Gα subunits are also substrates for tyrosine phosphorylation. A Gγ subunit is phosphorylated by protein kinase C, with the consequence that it interacts more tightly with a Gα subunit but less well with an effector.

Pathways of signal transduction employed by vertebrate Hedgehogs

Signalling by Hh (Hedgehog) proteins is among the most actively studied receptor-mediated phenomena relevant to development and post-embryonic homoeostatic events. The impact of signalling by the Hh proteins is profound, and work pertaining to the presentation of these proteins and the pathways engaged by them continues to yield unique insights into basic aspects of morphogenic signalling. We review here the mechanisms of signalling relevant to the actions of Hh proteins in vertebrates. We emphasize findings within the past several years on the recognition of, in particular, Sonic hedgehog by target cells, pathways of transduction employed by the seven-pass transmembrane protein Smoothened and end points of action, as manifest in the regulation of the Gli transcription factors. Topics of extended interest are those regarding the employment of heterotrimeric G-proteins and G-protein-coupled receptor kinases by Smoothened. We also address the pathways, insofar as known, linking Smoothened to the expression and stability of Gli1, Gli2 and Gli3. The mechanisms by which Hh proteins signal have few, if any, parallels. It is becoming clear in vertebrates, however, that several facets of signalling are shared in common with other venues of signalling. The challenge in understanding both the actions of Hh proteins and the overlapping forms of regulation will be in understanding, in molecular terms, both common and divergent signalling events.

Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors

Pathways of transduction employed by receptors for sphingosine 1-phosphate (S1P) are identified by the nature of second messengers and/or downstream targets regulated and, more formally, by direct assays of heterotrimeric G protein activation. The different methods generally agree. S1P1 couples to members of the Gi family, apparently selectively, although reported pertussis toxin (PTX)-insensitive actions make categorical statements regarding exclusivity difficult. S1P2 and S1P3 couple to members of the Gi, Gq, and G12/13 families. S1P4 couples to Gi and possibly G12/13, while S1P5 couples to Gi and G12/13 but not to Gq. In virtually all circumstances, coupling of S1P receptors to Gi is reflected in PTX-sensitive inhibition of adenylyl cyclase, activation of extracellular-regulated kinases (ERKs), and, depending on the cell, activation of phospholipase C (PLC). Coupling to Gq is reflected in PTX-insensitive activation of phospholipase C. Coupling to G12/13 is reflected in activation of Rho and subsequent activation of serum response factor (SRF). Specific linkages have been verified in almost all instances by receptor-promoted [35S]GTPgammaS/GDP exchange on identified G proteins.

Lysophosphatidic Acid Activates NF-κB in Fibroblasts A REQUIREMENT FOR MULTIPLE INPUTS

Lysophosphatidic acid (LPA) is a growth factor that exerts a number of well characterized biological actions on fibroblasts and other cells. In the present study, we investigated the possibility that LPA activates the transcription factor NF-κB. NF-κB is a target of cytokines, but its activation by other classes of agonists has raised considerable interest in the control of processes such as inflammation and wound healing through varied mechanisms. We find that LPA causes a marked activation of NF-κB in Swiss 3T3 fibroblasts as determined by the degradation of IκB-α in the cytosol and the emergence of κB binding activity in nuclear extracts. The EC50 for activation of NF-κB is 1–5 μm, a range similar to that reported for reinitiation of DNA synthesis and activation of the serum response element. Activation of NF-κB is attenuated by pertussis toxin and inhibitors of protein kinase C, and it is completely blocked by the Ca2+ chelator BAPTA-AM. The combination of phorbol ester and thapsigargin promotes an activation comparable with that of LPA. Activation by LPA is additionally inhibited by tyrphostin A25 but not genistein or AG1478, indicating a selective utilization of protein-tyrosine kinases, and by certain antioxidants, implying a role for reactive oxygen species. The activation is also inhibited by tricyclodecan-9-yl-xanthogenate (D609), implying a requirement for hydrolysis of phosphatidylcholine. The data demonstrate the utilization of multiple pathways in the activation of NF-κB by LPA, not inconsistent with the relevance of several families of GTP-binding regulatory proteins.

Identification of a G-protein in depolarizing rod bipolar cells

Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including Gt (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin. All of these antibodies reacted strongly with rods, but none reacted with bipolar cells. Antibodies to a different G-protein, G(o), reacted strongly with rod bipolar cells of three mammalian species (which are depolarizing and APB-sensitive). Also stained were subpopulations of cone bipolar cells but not the major depolarizing type in cat (b1). G(o) antibody also stained certain salamander bipolar cells. Thus, across a wide range of species, G(o) is present in retinal bipolar cells, and at least some of these are depolarizing and APB-sensitive.

The α Subunits of Gz and Gi Interact with the eyes absent Transcription Cofactor Eya2, Preventing Its Interaction with the Six Class of Homeodomain-containing Proteins

Yeast two-hybrid techniques were used to identify possible effectors for the heterotrimeric G protein Gz in human bone marrow cells. Eya2, a human homologue of the Drosophila Eya transcription co-activator, was identified. Eya2 interacts with activated Gαz and at least one other member of the Gαi family, Gαi2. Interactions were confirmed in mammalian two-hybrid and glutathione S-transferase fusion protein pull-down assays. Regions of Eya2-mediating interaction were mapped to the C-terminal Eya consensus domain. Eya2 is an intrinsically cytosolic protein that is translocated to the nucleus by members of the Six homeodomain-containing family of proteins. Activated Gαzand Gαi2 prevent Eya2 translocation and inhibit Six/Eya2-mediated activation of a reporter gene controlled through the MEF3/TATA promoter. Although G proteins are known to regulate the activity of numerous transcription factors, this regulation is normally achieved indirectly via one or more intermediates. We show here a novel functional regulation of a co-activator directly by G protein subunits.

Sonic Hedgehog Activates the GTPases Rac1 and RhoA in a Gli-Independent Manner Through Coupling of Smoothened to Gi Proteins

Smoothened signals through small G proteins in one type of noncanonical Hedgehog signaling. The vertebrate Hedgehog (Hh) pathway has essential functions during development and tissue homeostasis in normal physiology, and its dysregulation is a common theme in cancer. The Hh ligands (Sonic Hh, Indian Hh, and Desert Hh) bind to the receptors Patched1 and Patched2, resulting in inhibition of their repressive effect on Smoothened (Smo). Smo is a seven-transmembrane protein, which was only recently shown to function as a G protein–coupled receptor (GPCR) with specificity toward the heterotrimeric guanine nucleotide-binding protein Gi. In addition to activating Gi, Smo signals through its C-terminal tail to inhibit Suppressor of Fused, resulting in stabilization and activation of the Gli family of transcription factors, which execute a transcriptional response to so-called “canonical Hh signaling.” In this Presentation, we illustrate two outcomes of Hh signaling that are independent of Gli transcriptional activity and, thus, are defined as “noncanonical.” One outcome is dependent on Smo coupling to Gi proteins and exerts changes to the actin cytoskeleton through stimulation of the small guanosine triphosphatases (GTPases) RhoA and Rac1. These cytoskeletal changes promote migration in fibroblasts and tubulogenesis in endothelial cells. Signaling through the other noncanonical Hh pathway is independent of Smo and inhibits Patched1-induced cell death.

Regulation of Gαi Palmitoylation by Activation of the 5-Hydroxytryptamine-1A Receptor

Nearly all α subunits of heterotrimeric GTP-binding regulatory proteins (G proteins) are palmitoylated at cysteine residues near the N terminus. A regulated cycle of palmitoylation could provide a mechanism for modulating G protein signaling by affecting protein interactions and localization of the subunit. In the present studies we utilized both [3H]palmitate incorporation and pulse-chase techniques to address the dynamics of αi palmitoylation in Chinese hamster ovary cells. Both techniques demonstrated a dose- and time-dependent change in [3H]palmitate labeling of αi upon activation of stably expressed 5-hydroxytryptamine-1A receptors by the agonist (+/−)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (DPAT), with an EC50 of ∼10 nm. For the incorporation assay, DPAT elicited an approximate doubling in labeling at the earliest time point measured. For the pulse-chase assay, DPAT promoted a significant loss of radiolabel almost equally as fast. These data demonstrate that the exchange of palmitate on αi is increased upon stimulation of 5-hydroxytryptamine-1A receptors through the combined processes of depalmitoylation and palmitoylation. These results provide the basis for extending the concept of regulated exchange of palmitate beyond Gs and provide a framework for exploring the specific functional attributes of the palmitoylated and depalmitoylated forms of subunit.

Agonist Receptors and G proteins as Mediators of Platelet Activation

Recent studies have helped to define the earliest events of signal transduction in platelets, particularly those involved in the generation of second messengers. The best-understood of these events are those which involve guanine nucleotide binding regulatory proteins. G proteins are heterotrimers comprised of alpha, beta and gamma subunits, each of which can exist in multiple forms. Some, but not all, of the known variants of G alpha are substrates for ADP-ribosylation by pertussis toxin, a modification which disrupts the flow of information from receptor to effector. The G proteins that have been identified in platelets to date are Gs, Gi1, Gi2, Gi3, Gz and Gq. Gs and one or more of the Gi family members regulate cAMP formation by adenylylcyclase. Gi may also be responsible for the pertussis toxin-sensitive activation of phospholipase C which occurs when platelets are activated by thrombin. Gq is thought to be responsible for the pertussis toxin-resistant activation of phospholipase C by TxA2. Gz does not have an established role, but has the unique property of being phosphorylated by protein kinase C during platelet activation. Recent efforts to clone the receptors that interact with G proteins in platelets have been successful for epinephrine, thrombin, TxA2 and platelet activating factor. Each of these resembles other G protein-coupled receptors, being comprised of a single polypeptide with 7 transmembrane domains. In the case of thrombin, receptor activation is thought to involve a unique mechanism in which thrombin cleaves its receptor, creating a new N-terminus that can serve as a tethered ligand. Peptides corresponding to the tethered ligand can mimic the effects of thrombin, while antibodies to the same domain inhibit platelet activation. Shortly after activation, thrombin receptors become resistant to re-activation by thrombin. This desensitization, which appears to be due to a combination of proteolysis, phosphorylation and internalization, provides a potential mechanism for limiting the duration of thrombin-initiated signals in platelets.