Activation of a Frizzled-2/β-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Gαo and Gαt

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven-transmembrane-spanning motif characteristic of G protein-linked receptors, though no loss-of-function studies have demonstrated a requirement for G proteins for Frizzled signaling. We engineered a Frizzled-2 chimera responsive to β-adrenergic agonist by using the ligand-binding domains of the β2-adrenergic receptor. The expectation was that the chimera would be sensitive both to drug-mediated activation and blockade, thereby circumventing the problem of purifying soluble and active Wnt ligand to activate Frizzled. Expression of the chimera in zebrafish embryos demonstrated isoproterenol (ISO)-stimulated, propranolol-sensitive calcium transients, thereby confirming the β-adrenergic nature of Wnt signaling by the chimeric receptor. Because F9 embryonic teratocarcinoma cells form primitive endoderm after stable transfection of Frizzled-2 chimera and stimulation with ISO, they were subject to depletion of G protein subunits. ISO stimulation of endoderm formation of F9 stem cells expressing the chimeric receptor was blocked by pertussis toxin and by oligodeoxynucleotide antisense to Gαo, Gαt2, and Gβ2. Our results demonstrate the requirement of two pertussis toxin-sensitive G proteins, Gαo and Gαt, for signaling by the Frizzled-2 receptor.

Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): Evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry

Homologues of Drosophilia transient receptor potential (TRP) have been proposed to be unitary subunits of plasma membrane ion channels that are activated as a consequence of active or passive depletion of Ca2+ stores. In agreement with this hypothesis, cells expressing TRPs display novel Ca2+-permeable cation channels that can be activated by the inositol 1,4,5-trisphosphate receptor (IP3R) protein. Expression of TRPs alters cells in many ways, including up-regulation of IP3Rs not coded for by TRP genes, and proof that TRP forms channels of these and other cells is still missing. Here, we document physical interaction of TRP and IP3R by coimmunoprecipitation and glutathione S-transferase-pulldown experiments and identify two regions of IP3R, F2q and F2g, that interact with one region of TRP, C7. These interacting regions were expressed in cells with an unmodified complement of TRPs and IP3Rs to study their effect on agonist- as well as store depletion-induced Ca2+ entry and to test for a role of their respective binding partners in Ca2+ entry. C7 and an F2q-containing fragment of IP3R decreased both forms of Ca2+ entry. In contrast, F2g enhanced the two forms of Ca2+ entry. We conclude that store depletion-activated Ca2+ entry occurs through channels that have TRPs as one of their normal structural components, and that these channels are directly activated by IP3Rs. IP3Rs, therefore, have the dual role of releasing Ca2+ from stores and activating Ca2+ influx in response to either increasing IP3 or decreasing luminal Ca2+.

Construction of a high-affinity receptor site for dihydropyridine agonists and antagonists by single amino acid substitutions in a non-l-type Ca2+ channel

The activity of l-type Ca2+ channels is increased by dihydropyridine (DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore-forming α1 subunits of l-type Ca2+ channels. To define the minimal requirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive l-type α1 subunits into the S5 and S6 transmembrane segments of domain III and the S6 transmembrane segment of domain IV of the DHP-insensitive P/Q-type α1A subunit. The resulting chimeric α1A/DHPS subunit bound DHP antagonists with high affinity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of these nine amino acid residues yielded 86% of the binding energy of the l-type α1C subunit and 92% of the binding energy of the l-type α1S subunit for the high-affinity DHP antagonist PN200–110. The activity of chimeric Ca2+ channels containing α1A/DHPS was increased 3.5 ± 0.7-fold by the DHP agonist (−)Bay K8644. The effect of this agonist was stereoselective as in l-type Ca2+ channels since (+) Bay K8644 inhibited the activity of α1A/DHPS. The results show conclusively that DHP agonists and antagonists bind to a single receptor site at which they have opposite effects on Ca2+ channel activity. This site contains essential components from both domains III and IV, consistent with a domain interface model for binding and allosteric modulation of Ca2+ channel activity by DHPs.

The Thrombospondin Receptor CD47 (IAP) Modulates and Associates with α2β1 Integrin in Vascular Smooth Muscle Cells

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a β1 integrin, rather than αvβ3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to α2 and β1 integrin subunits and to IAP. A combination of antiα2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antiαvβ3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between α2β1 and IAP was detected by the coimmunoprecipitation of both α2 and β1 integrin subunits with IAP. These data suggest that IAP can associate with α2β1 integrin and modulate its function.

Activation of RhoA by Lysophosphatidic Acid and Gα12/13 Subunits in Neuronal Cells: Induction of Neurite Retraction

Neuronal cells undergo rapid growth cone collapse, neurite retraction, and cell rounding in response to certain G protein–coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable assay. Furthermore, the nature of the G protein(s) mediating LPA-induced neurite retraction remains unknown. We have developed a Rho activation assay that is based on the specific binding of active RhoA to its downstream effector Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of ROK pulls down activated but not inactive RhoA from cell lysates. Using GST-ROK, we show that in N1E-115 neuronal cells LPA activates endogenous RhoA within 30 s, concomitant with growth cone collapse. Maximal activation occurs after 3 min when neurite retraction is complete and the actin cytoskeleton is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated Gα12 and Gα13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a G12/13-initiated pathway that involves protein-tyrosine kinase activity.

Division of Labor among the α6β4 Integrin, β1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and β-Casein Expression in Mammary Epithelial Cells

Contact of cultured mammary epithelial cells with the basement membrane protein laminin induces multiple responses, including cell shape changes, growth arrest, and, in the presence of prolactin, transcription of the milk protein β-casein. We sought to identify the specific laminin receptor(s) mediating the multiple cell responses to laminin. Using assays with clonal mammary epithelial cells, we reveal distinct functions for the α6β4 integrin, β1 integrins, and an E3 laminin receptor. Signals from laminin for β-casein expression were inhibited in the presence of function-blocking antibodies against both the α6 and β1 integrin subunits and by the laminin E3 fragment. The α6-blocking antibody perturbed signals mediated by the α6β4 integrin, and the β1-blocking antibody perturbed signals mediated by another integrin, the α subunit(s) of which remains to be determined. Neither α6- nor β1-blocking antibodies perturbed the cell shape changes resulting from cell exposure to laminin. However, the E3 laminin fragment and heparin both inhibited cell shape changes induced by laminin, thereby implicating an E3 laminin receptor in this function. These results elucidate the multiplicity of cell-extracellular matrix interactions required to integrate cell structure and signaling and ultimately permit normal cell function.

Selection of Gβ Subunits with Point Mutations That Fail to Activate Specific Signaling Pathways In Vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G Protein in Dictyostelium discoideum

In Dictyostelium discoideum, a unique Gβ subunit is required for a G protein–coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Gα2, and Gβ genes completely impair all these responses. To dissect specificity in Gβγ signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gβ genes and isolated Gβ alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gβ subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gβγ by making point mutations on Gβ. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gβs and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtβγ interacts with its regulators, Gα and phosducin.

Role of Syk in B-cell development and antigen-receptor signaling

Antigen receptors (BCRs) on developing B lymphocytes play two opposing roles—promoting survival of cells that may later bind a foreign antigen and inhibiting survival of cells that bind too strongly to self-antigens. It is not known how these opposing outcomes are signaled by BCRs on immature B cells. Here we analyze the effect of a null mutation in the Syk tyrosine kinase on maturing B cells displaying a transgene-encoded BCR that binds hen egg lysozyme (HEL). In the absence of HEL antigen, HEL-specific BCRs are expressed normally on the surface of Syk-deficient immature B-lineage cells, but this fails to promote maturation beyond the earliest stages of B-lineage commitment. Binding of HEL antigen, nevertheless, triggers phosphorylation of CD79α/β BCR subunits and modulation of receptors from the surface in Syk-deficient cells, but it cannot induce an intracellular calcium response. Continuous binding of low- or high-avidity forms of HEL, expressed as self-antigens, fails to restore the signal needed for maturation. Compared with the effects in the same system of null mutations in other BCR signaling elements, such as CD45 and Lyn kinase, these results indicate that Syk is essential for transmitting a signal that initiates the program of B-lymphocyte maturation.

The γ-aminobutyric acid type A receptor (GABAAR)-associated protein GABARAP interacts with gephyrin but is not involved in receptor anchoring at the synapse

γ-Aminobutyric acid type A receptors (GABAARs) are ligand-gated chloride channels that exist in numerous distinct subunit combinations. At postsynaptic membrane specializations, different GABAAR isoforms colocalize with the tubulin-binding protein gephyrin. However, direct interactions of GABAAR subunits with gephyrin have not been reported. Recently, the GABAAR-associated protein GABARAP was found to bind to the γ2 subunit of GABAARs. Here we show that GABARAP interacts with gephyrin in both biochemical assays and transfected cells. Confocal analysis of neurons derived from wild-type and gephyrin-knockout mice revealed that GABARAP is highly enriched in intracellular compartments, but not at gephyrin-positive postsynaptic membrane specializations. Our data indicate that GABARAP–gephyrin interactions are not important for postsynaptic GABAAR anchoring but may be implicated in receptor sorting and/or targeting mechanisms. Consistent with this idea, a close homolog of GABARAP, p16, has been found to function as a late-acting intra-Golgi transport factor.

Nerve growth factor rapidly suppresses basal, NMDA-evoked, and AMPA-evoked nitric oxide synthase activity in rat hippocampus in vivo

In adult forebrain, nerve growth factor (NGF) influences neuronal maintenance and axon sprouting and is neuroprotective in several injury models through mechanisms that are incompletely understood. Most NGF signaling is thought to occur after internalization and retrograde transport of trkA receptor and be mediated through the nucleus. However, NGF expression in hippocampus is rapidly and sensitively regulated by synaptic activity, suggesting that NGF exerts local effects more dynamically than possible through signaling requiring retrograde transport to distant afferent neurons. Interactions have been reported between NGF and nitric oxide (NO). Because NO affects both neural plasticity and degeneration, and trk receptors can mediate signaling within minutes, we hypothesized that NGF might rapidly modulate NO production. Using in vivo microdialysis we measured conversion of l-[14C]arginine to l-[14C]citrulline as an accurate reflection of NO synthase (NOS) activity in adult rat hippocampus. NGF significantly reduced NOS activity to 61% of basal levels within 20 min of onset of delivery and maintained NOS activity at less than 50% of baseline throughout 3 hr of delivery. This effect did not occur with control protein (cytochrome c) and was not mediated by an effect of NGF on glutamate levels. In addition, simultaneous delivery of NGF prevented significant increases in NOS activity triggered by the glutamate receptor agonists N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Rapid suppression by NGF of basal and glutamate-stimulated NOS activity may regulate neuromodulatory functions of NO or protect neurons from NO toxicity and suggests a novel mechanism for rapidly mediating functions of NGF and other neurotrophins.

Two functionally dependent acetylcholine subunits are encoded in a single Caenorhabditis elegans operon

The deg-3 gene from the nematode Caenorhabditis elegans encodes an α subunit of a nicotinic acetylcholine receptor that was first identified by a dominant allele, u662, which produced neuronal degeneration. Because deg-3 cDNAs contain the SL2 trans-spliced leader, we suggested that deg-3 was transcribed as part of a C. elegans operon. Here we show that des-2, a gene in which mutations suppress deg-3(u662), is the upstream gene in that operon. The des-2 gene also encodes an α subunit of a nicotinic acetylcholine receptor. As expected for genes whose mRNAs are formed from a single transcript, both genes have similar expression patterns. This coexpression is functionally important because (i) des-2 is needed for the deg-3(u662) degenerations in vivo; (ii) an acetylcholine-gated channel is formed in Xenopus oocytes when both subunits are expressed but not when either is expressed alone; and (iii) channel activity, albeit apparently altered from that of the wild-type channel, results from the expression of a u662-type mutant subunit but, again, only when the wild-type DES-2 subunit is present. Thus, the operon structure appears to regulate the coordinate expression of two channel subunits.

Synaptic and extrasynaptic γ-aminobutyric acid type A receptor clusters in rat hippocampal cultures during development

We have simultaneously measured the expression of postsynaptic γ-aminobutyric acid type A (GABAA) receptor clusters and of presynaptic boutons in neonatal rat hippocampal cultures between days 1 and 30. GABAA receptors were labeled with antibodies recognizing the extracellular domains of β2/3 and γ2 subunits. Boutons were visualized by activity-dependent uptake of the styryl dye FM4-64, or by antibodies against the presynaptic vesicular protein SV2 or the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). GABAA receptor clusters could be seen in living neurons already 6 h after culturing, much before presynaptic markers could be identified in nerve terminals. The densities of receptor clusters that contained the β2/3 subunits were constant between days 10 and 30 in culture, whereas γ2 subunit-containing clusters fluctuated and reached a maximum on day 20. SV2 and GAD staining could be measured from day 2 onwards. Clustering of GAD in presynaptic terminals and FM4-64 uptake were observed only at day 5 and afterward. SV2 staining and FM4-64 uptake increased in parallel between days 5 and 20 and remained constant thereafter. GAD-stained boutons were fewer than those labeled with other, less specific, presynaptic stains. They reached a maximum on day 20 and fell again toward day 30. Double labeling of GABAA receptors and of presynaptic boutons in neurons during differentiation showed that, even after 30 days in culture, large fractions of GABAA receptor clusters containing β2/3 and/or γ2 subunits remained extrasynaptic.

α10: A determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells

We report the cloning and characterization of rat α10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the α10 nAChR subunit gene is most similar to the rat α9 nAChR, and both α9 and α10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with α10 cRNA alone or in pairwise combinations with either α2-α6 or β2-β4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of α9 and α10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric α9 channels, the α9α10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current–voltage relationship, and a biphasic response to changes in extracellular Ca2+ ions. The pharmacological profiles of homomeric α9 and heteromeric α9α10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both α9 and α10 subunits.

Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus

Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [3H]ryanodine binding that was sensitive to activation by Ca2+ and caffeine and to inhibition by Mg2+. 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the “clamp” structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.

Enhanced tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate receptor in long-term potentiation.

Both serine/threonine and tyrosine phosphorylation of receptor proteins have been implicated in the process of long-term potentiation (LTP), but there has been no direct demonstration of a change in receptor phosphorylation after LTP induction. We show that, after induction of LTP in the dentate gyrus of anesthetized adult rats, there is an increase in the tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate (NMDA) receptor (NR2B), as well as several other unidentified proteins. Tyrosine phosphorylation of NR2B was measured in two ways: binding of antiphosphotyrosine antibodies (PY20) to glycoprotein(s) of 180 kDa (GP180) purified on Con A-Sepharose and binding of anti-NR2B antibodies to tyrosine-phosphorylated proteins purified on PY20-agarose. Three hours after LTP induction, anti-NR2B binding to tyrosine phosphorylated proteins, expressed as a ratio of tetanized to control dentate (Tet/Con), was 2.21 +/- 0.50 and PY20 binding to GP180 was 1.68 +/- 0.16. This increase in the number of tyrosine phosphorylated NR2B subunits occurred without a change in the total number of NR2B subunits. When the induction of LTP was blocked by pretreatment of the animal with the NMDA receptor antagonist MK801, the increase in PY20 binding to GP180 was also blocked (Tet/Con = 1.09 +/- 0.26). The increased PY20 binding to GP180 was also apparent 15 min after LTP induction (Tet/Con = 1.41 +/- 0.16) but not detectable 5 min after LTP induction (Tet/Con = 1.01 +/- 0.19). These results suggest that tyrosine phosphorylation of the NMDA receptor contributes to the maintenance of LTP.