Karsten Spicher

Roles of Gβγ in membrane recruitment and activation of p110γ/p101 phosphoinositide 3-kinase γ

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3–binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110γ/p101 PI3Kγ is activated by Gβγ on stimulation of G protein–coupled receptors. It is currently unknown whether in living cells Gβγ acts as a membrane anchor or an allosteric activator of PI3Kγ, and which role its noncatalytic p101 subunit plays in its activation by Gβγ. Using GFP-tagged PI3Kγ subunits expressed in HEK cells, we show that Gβγ recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein–mediated activation of PI3Kγ in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3–binding PH domains. Furthermore, membrane-targeted p110γ displayed basal enzymatic activity, but was further stimulated by Gβγ, even in the absence of p101. Therefore, we conclude that in vivo, Gβγ activates PI3Kγ by a mechanism assigning specific roles for both PI3Kγ subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of Gβγ with p110γ contributes to activation of PI3Kγ.

μ and δ opioid receptors differentially couple to G protein subtypes in membranes of human neuroblastoma SH-SY5Y cells

Abstract Opioids are regarded to act via receptors interacting with heterotrimeric pertussis toxin (PTX)-sensitive G proteins. In membranes of SH-SY5Y cells, the μ-selective agonist [d-Ala 2 ,N-Me-Phe 4 , Gly 5 -ol]-enkephalin (DAGO) and the δ-selective agonist [d-Pen 2 ,Pen 5 ]-enkephalin (DPDPE) stimulated incorporation of the photoreactive GTP analog [α- 32 P]GTP azidoanilide into proteins comigrating with the α subunits of G i1 , G i2 , G i3 , G o1 , and another form of G o , presumably G o2 . In membranes of PTX-treated cells, both agonists were ineffective. Subtype-specific immunoprecipitation of G protein α subunits photolabeled in the absence or presence of agonists revealed profound differences between μ and δ opioid receptors in coupling to PTX-sensitive G proteins. Whereas activated ° opioid receptors preferentially coupled to G i1 , activated μ opioid receptors more effectively coupled to G i3 . Additionally, we provide evidence that G o subtypes are also differentially activated by the two receptors. Thus, μ and δ opioid receptors appear to discriminate between PTX-sensitive G proteins and lead to activation of distinct G protein subtypes.

Macrophages Induce the Inflammatory Response in the Pulmonary Arthus Reaction through Gαi2 Activation That Controls C5aR and Fc Receptor Cooperation

Complement and FcγR effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory FcγRIIB, increased levels of activating FcγRIII, and highly induced FcγR-mediated TNF-α and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of Gi-type G protein signaling in C5aR-mediated control of the regulatory FcγR system in vitro, and analysis of the various C5aR-, FcγR-, and Gi-deficient mice verifies the importance of Gαi2-associated C5aR and the FcγRIII-FcγRIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and FcγRIII-positive cells into C5aR- and FcγRIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or FcγRIII do not possess any such inducibility of immune complex disease, whereas reconstitution with FcγRIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a Gαi2-dependent signal for modulating the two opposing FcγR, FcγRIIB and FcγRIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.

Most central nervous system D2 dopamine receptors are coupled to their effectors by Go

We reported previously that Go-deficient mice develop severe neurological defects that include hyperalgesia, a generalized tremor, lack of coordination, and a turning syndrome somewhat reminiscent of unilateral lesions of the dopaminergic nigro-striatal pathway. By using frozen coronal sections of serially sectioned brains of normal and Go-deficient mice, we studied the ability of several G protein coupled receptors to promote binding of GTPγS to G proteins and the ability of GTP to promote a shift in the affinity of D2 dopamine receptor for its physiologic agonist dopamine. We found a generalized, but not abolished reduction in agonist-stimulated binding of GTPγS to frozen brain sections, with no significant left–right differences. Unexpectedly, the ability of GTP to regulate the binding affinity of dopamine to D2 receptors (as seen in in situ [35S]sulpiride displacement curves) that was robust in control mice, was absent in Go-deficient mice. The data suggest that most of the effects of the Gi/Go-coupled D2 receptors in the central nervous system are mediated by Go instead of Gi1, Gi2, or Gi3. In agreement with this, the effect of GTP on dopamine binding to D2 receptors in double Gi1 plus Gi2- and Gi1 plus Gi3-deficient mice was essentially unaffected.

Gq and G11 are concurrently activated by bombesin and vasopressin in Swiss 3T3 cells.

The α‐subunits of the widely expressed G‐proteins Gq and G11 indistinguishably activate β‐isoforms of phospholipase C. In this report we have tested whether differences exist in the activation of both G‐proteins via phospholipase C‐linked receptors. We found that bombesin and vasopressin, with very similar potencies and time dependencies, induce the activation of both Gq and G11 in Swiss 3T3 cells, suggesting that these G‐proteins, at least in part, serve interchangeable functions.

The α‐subunits of G‐proteins G12 and G13 are palmitoylated, but not amidically myristoylated

The α‐subunits of the G‐proteins G12 and G13, were expressed with a baculovirus system in insect cells and analysed for acylation. Both proteins incorporated tritiated palmitic and to a lesser extent also tritiated myristic acid. Radiolabel from both fatty acids was sensitive to treatment with neutral hydroxylamine. This result supports a thioester‐type fatty acid bond and argues against amidical N‐myristoylation. Fatty acid analysis after labeling with [3H]palmitic acid showed that palmitate represents the predominant fatty acid linked to Gα12 and Gα13. Separation of cells into cytosolic and membranous fractions revealed that palmitoylated α‐subunits of G12 were exclusively membrane‐bound, whereas [35S]methionine‐labeled proteins were detected in soluble and particulate fractions. Inhibition of protein synthesis with cycloheximide did not block palmitoylation of the α‐subunits. which indicates that palmitoylation occurs independently of protein synthesis.

Inhibition of voltage-dependent Ca2+ channels via alpha 2-adrenergic and opioid receptors in cultured bovine adrenal chromaffin cells.

Adrenal chromaffin cells secrete catecholamindes and opioids. The effects of these agents on whole-cell Ca2+ channel currents were studied, using bovine adrenal chromaffin cells kept in short term culture. Ca2+ channel currents recorded during voltageclamp pulses from a holding potential of −80 mV to 0 mV were reversibly reduced by 10 μM epinephrine (in the presence of 1 μM propranolol) or 5 μM of the synthetic opioid, d-Ala2-d-Leu5-enkephalin (DADLE) by approximately 35% and 25%, respectively. The inhibitory action of epinephrine was mimicked by clonidine, reduced by yohimbine but not affected by prazosin. The DADLE-induced reduction of the Ca2+ channel current was antagonized by naloxone. The dihydropyridine (+)PN 200-110 (5 μM) reduced the Ca2+ channel current by approximately 40%; the Ca2+ channel current inhibited by (+)PN 200-110 was not further reduced by epinephrine. Intracellular infusion of guanosine-5′-O-(2-thiodiphosphate) and pretreatment of cells with pertussis toxin abolished the inhibitory effect of both epinephrine and DADLE. In membranes of adrenal chromaffin cells, four pertussis-toxin-sensitive G-proteins were identified, including Gi1, Gi2, Go1 and another Go subtype, possibly Go2. The data show that activation of α2-adrenergic and opioid receptors causes an inhibition of dihydropyridine-sensitive Ca2+ channels in adrenal chromaffm cells. These inhibitory modulations are mediated by pertussis-toxin-sensitive G-proteins and may represent a mechanism for a negative feedback signal by agents released from the adrenal medulla.

A high-affinity bradykinin receptor in membranes from rat myometrium is coupled to pertussis toxin-sensitive G-proteins of the Gi family.

In rat myometrial membranes, two 3H-Bradykinin binding sites with KD values of 16 pM and 1.0 nM were identified. Employed at pM concentrations, bradykinin stimulated high affinity GTPases. This effect was abolished by the bradykinin antagonist, [D-Arg(Hyp3-Thi5,8, D-Phe7)]bradykinin (10 microM), and by treatment of membranes with pertussis toxin. Myometrial membranes contained two pertussis toxin substrates of 40 and 41 kDa, which corresponded immunologically to alpha-subunits of Gi-type G-proteins. The faster migrating substrate was tentatively identified as Gi2 alpha-subunit. The electrophoretic mobility of the slower migrating Gi alpha-subunit was very similar to that of the Gi3 alpha-subunit. Go alpha-subunits were not detected. Thus, in uterine smooth muscle, G-proteins of the Gi-family (Gi2, Gi3) couple high-affinity bradykinin receptors to their effector enzymes.

Agonist-sensitive binding of a photoreactive GTP analog to a G-protein α-subunit in membranes of HL-60 cells

Myeloid‐differentiated HL‐60 cells were used to study the activation of G‐proteins by receptor agonists. Following incubation of membranes with the photoreactive GTP analog. [α‐32P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the Gi2 α‐subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N‐ionnyl‐methionyl‐leucyi‐phenylalanin (FMLP), absolutely required Mg2+; FMLP stimulated photolabeling at all Mg2+ concentrations employed (up to 30 mM). Addition of GDP (3–50 μM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B4 and C5a also stimulated photolabeling of 40 kDa proteins. The results indicate that (i) the major G‐protein in HL‐60 cells, Gi2 requires Mg2+ for basal and receptor‐stimulated activity, (ii) effective receptor‐mediated activation of G‐proteins is observed at mM concentrations of Mg2+, and (iii) receptor agonists apparently reduce the affinity of G‐proteins for GDP.

Adipocyte plasma membranes contain two Gi subtypes but are devoid of Go.

Antisera generated against synthetic peptides were used to identify G‐protein α‐subunits in plasma membranes from rat adipocytes. Applying the immunoblot technique, we detected two Gs α‐subunits of 42 and 43 kDa, corresponding to the two cholera toxin substrates, and two Gi α‐subunits of 40 and 41 kDa, corresponding to the two pertussis toxin substrates present in these membranes. The 40 kDa protein was tentatively identified as the Gi2, α‐subunit. A serum specific for the Go α‐subunit failed to detect any immunoreactive protein. Thus plasma membranes of adipocytes possess two forms of Gi but not Go.