James C. Garrison

Much Ado about Adenosine: Adenosine Synthesis and Function in Regulatory T Cell Biology

Recent studies have reported that adenosine is a significant mediator of regulatory T cell (Treg) function. Indeed, activation of the adenosine receptor subtypes expressed by a broad range of immune and inflammatory cells attenuates inflammation in several disease models. This anti-inflammatory response is associated with an increase in intracellular cAMP that inhibits cytokine responses of many immune/inflammatory cells, including T cells and APCs. Thus, adenosine produced by Tregs can provide a paracrine feedback that shapes the host response following an immunologic provocation. This review discusses the evidence that adenosine is an integral part of Treg biology and presents some of the mechanisms that may account for its contribution to the resolution of inflammation and the regulation of the immune/inflammatory cell phenotype.

T-type calcium channel regulation by specific G-protein βγ subunits

Low-voltage-activated (LVA) T-type calcium channels have a wide tissue distribution and have well-documented roles in the control of action potential burst generation and hormone secretion. In neurons of the central nervous system and secretory cells of the adrenal and pituitary, LVA channels are inhibited by activation of G-protein-coupled receptors that generate membrane-delimited signals, yet these signals have not been identified. Here we show that the inhibition of α1H (Cav3.2), but not α1G (Cav3.1) LVA Ca2+ channels is mediated selectively by β2γ2 subunits that bind to the intracellular loop connecting channel transmembrane domains II and III. This region of the α1H channel is crucial for inhibition, because its replacement abrogates inhibition and its transfer to non-modulated α1G channels confers β2γ2-dependent inhibition. βγ reduces channel activity independent of voltage, a mechanism distinct from the established βγ-dependent inhibition of non-L-type high-voltage-activated channels of the Cav2 family. These studies identify the α1H channel as a new effector for G-protein βγ subunits, and highlight the selective signalling roles available for particular βγ combinations.

Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors.

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein α subunits and by the composition of the βγ dimers. To determine the role of the γ subunit prenyl modification in this interaction, the CaaX motifs in the γ1 and γ2 subunits were altered to direct modification with different prenyl groups, recombinant βγ dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the βγ dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the α subunit. The β1γ1 dimer (modified with farnesyl) was significantly less effective than β1γ2 (modified with geranylgeranyl) in either assay. The β1γ1-S74L dimer (modified with geranylgeranyl) was nearly as effective as β1γ2 in either assay. The β1γ2-L71S dimer (modified with farnesyl) was significantly less active than β1γ2. Using 125I-labeled βγ subunits, it was determined that native and altered βγ dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the γ subunit is an important determinant of the interaction between receptors and G protein γ subunits.

Tubulin, Gq, and Phosphatidylinositol 4,5-Bisphosphate Interact to Regulate Phospholipase Cβ1 Signaling

The cytoskeletal protein, tubulin, has been shown to regulate adenylyl cyclase activity through its interaction with the specific G protein α subunits, Gαs or Gαi1. Tubulin activates these G proteins by transferring GTP and stabilizing the active nucleotide-bound Gα conformation. To study the possibility of tubulin involvement in Gαq-mediated phospholipase Cβ1 (PLCβ1) signaling, the m1 muscarinic receptor, Gαq, and PLCβ1 were expressed in Sf9 cells. A unique ability of tubulin to regulate PLCβ1 was observed. Low concentrations of tubulin, with guanine nucleotide bound, activated PLCβ1, whereas higher concentrations inhibited the enzyme. Interaction of tubulin with both Gαq and PLCβ1, accompanied by guanine nucleotide transfer from tubulin to Gαq, is suggested as a mechanism for the enzyme activation. The PLCβ1 substrate, phosphatidylinositol 4,5-bisphosphate, bound to tubulin and prevented microtubule assembly. This observation suggested a mechanism for the inhibition of PLCβ1 by tubulin, since high tubulin concentrations might prevent the access of PLCβ1 to its substrate. Activation of m1 muscarinic receptors by carbachol relaxed this inhibition, probably by increasing the affinity of Gαq for tubulin. Involvement of tubulin in the articulation between PLCβ1 signaling and microtubule assembly might prove important for the intracellular governing of a broad range of cellular events.

The G Protein β5 Subunit Interacts Selectively with the Gq α Subunit

The diversity in the heterotrimeric G protein α, β, and γ subunits may allow selective protein-protein interactions and provide specificity for signaling pathways. We examined the ability of five α subunits (αi1, αi2, αo, αs, and αq) to associate with three β subunits (β1, β2, and β5) dimerized to a γ2 subunit containing an amino-terminal hexahistidine-FLAG affinity tag (γ2HF). Sf9 insect cells were used to overexpress the recombinant proteins. The hexahistidine-FLAG sequence does not hinder the function of the β1γ2HF dimer as it can be specifically eluted from an αi1-agarose column with GDP and AlF4 −, and purified β1γ2HF dimer stimulates type II adenylyl cyclase. The β1γ2HF and β2γ2HF dimers immobilized on an anti-FLAG affinity column bound all five α subunits tested, whereas the β5γ2HF dimer bound only αq. The ability of other α subunits to compete with the αqsubunit for binding to the β5γ2HF dimer was tested. Addition of increasing amounts of purified, recombinant αi1 to the αq in a Sf9 cell extract did not decrease the amount of αq bound to the β5γ2HF column. When G proteins in an extract of brain membranes were activated with GDP and AlF4 − and deactivated in the presence of equal amounts of the β1γ2HF or β5γ2HF dimers, only αq bound to the β5γ2HF dimer. The αq-β5γ2HF interaction on the column was functional as GDP, and AlF4 −specifically eluted αq from the column. These results indicate that although the β1 and β2 subunits interact with α subunits from the αi, αs, and αq families, the structurally divergent β5 subunit only interacts with αq.

Phosphorylation of P-Rex1 by the cyclic amp-dependent protein kinase inhibits the phosphatidylinositiol (3,4,5)-trisphosphate and Gβγ-mediated regulation of its activity

Rac activation is a key step in chemotaxis of hematopoietic cells, which is both positively and negatively regulated by receptors coupled to heterotrimeric G proteins. P-Rex1, a Rac-specific guanine nucleotide exchange factor, is dually activated by phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the Gβγ subunits of heterotrimeric G proteins. This study explored the regulation of P-Rex1 by phosphorylation with the cAMP-dependent protein kinase (protein kinase A) in vitro and by Gi- and Gs-coupled receptors in HEK293T cells. P-Rex1 isolated from Sf9 and HEK293T cells migrates as two distinct bands that are partially phosphorylated. Phosphorylation of P-Rex1 with protein kinase A (PKA) inhibits the PIP3- and Gβγ-stimulated P-Rex1 guanine nucleotide exchange activity on Rac. The guanine nucleotide exchange factor activity of three different forms of P-Rex1 (native Sf9, de-phosphorylated, and phosphorylated) was examined in the presence of PIP3 and varying concentrations of Gβ1γ2. Gβ1γ2 was 47-fold less potent in activating the phosphorylated form of P-Rex1 compared with the de-phosphorylated form. HEK293T cells expressing P-Rex1 were labeled with 32P and stimulated with lysophosphatidic acid (LPA) to release Gβγ or isoproterenol to activate PKA. Treatment with isoproterenol or Sp-cAMPS, a potent activator of PKA, increased the incorporation of 32P into P-Rex1. LPA increased the amount of GTP-bound Rac in the cells and isoproterenol reduced basal levels of GTP-bound Rac and blunted the effect of LPA. Treatment of the cells with Sp-cAMPS also reduced the levels of GTP-bound Rac. These results outline a novel mechanism for Gs-linked receptors to regulate the function of P-Rex1 and inhibit its function in cells.

Endothelium-derived relaxing factor release associated with increased endothelial cell inositol trisphosphate and intracellular calcium

The release of eicosanoids and endothelium-derived relaxing factor (EDRF) from endothelial cells is thought to involve a calcium-dependent step. Using cultured bovine aortic endothelial cells as a model system, we have examined the relation between agonist-induced changes in inositol polyphosphates and calcium levels within the endothelial cells and extracellular calcium on EDRF release. In a superfusion-cascade system, EDRF was detected by the relaxation of a rabbit aortic ring without endothelium suspended beneath a column of cultured endothelial cells. Endothelial cell stimulation by bradykinin or melittin induced dose-dependent relaxation of the bioassay ring. In addition, bradykinin and melittin stimulated an increase in intracellular calcium concentration in fura-2 loaded endothelial cells and an increase in inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) in cells prelabeled with 3H-myoinositol. Bradykinin stimulation produced transient increases in Ins(1,4,5)P3, fura-2 fluorescence and transient EDRF release. Melittin stimulation induced more prolonged release of EDRF from the endothelial cell column, which was correlated with sustained increases in the fura-2 signal and the level of Ins(1,4,5)P3. Omission of calcium from the cell superfusate attenuated, but did not eliminate, bradykinin-induced EDRF release and the calcium transient, whereas the melittin-induced responses were only slightly attenuated. Endothelial cells clearly demonstrate receptor-activation of phospholipase C and release of sequestered calcium from subcellular sites in response to Ins(1,4,5)P3. These results imply that EDRF release is correlated with increased intracellular calcium levels seen in the absence of extracellular calcium. However, sustained release of EDRF does require influx of extracellular calcium via an undefined mechanism.

Phosphorylation of the G Protein γ12 Subunit Regulates Effector Specificity

Although the G protein βγ dimer is an important mediator in cell signaling, the mechanisms regulating its activity have not been widely investigated. The γ12subunit is a known substrate for protein kinase C, suggesting phosphorylation as a potential regulatory mechanism. Therefore, recombinant β1γ12 dimers were overexpressed using the baculovirus/Sf9 insect cell system, purified, and phosphorylated stoichiometrically with protein kinase C α. Their ability to support coupling of the Gi1 α subunit to the A1 adenosine receptor and to activate type II adenylyl cyclase or phospholipase C-β was examined. Phosphorylation of the β1γ12 dimer increased its potency in the receptor coupling assay from 6.4 to 1 nm, changed theK act for stimulation of type II adenylyl cyclase from 14 to 37 nm, and decreased its maximal efficacy by 50%. In contrast, phosphorylation of the dimer had no effect on its ability to activate phospholipase C-β. The native β1γ10 dimer, which has 4 similar amino acids in the phosphorylation site at the N terminus, was not phosphorylated by protein kinase C α. Creation of a phosphorylation site in the N terminus of the protein (Gly4 → Lys) resulted in a β1γ10G4K dimer which could be phosphorylated. The activities of this βγ dimer were similar to those of the phosphorylated β1γ12 dimer. Thus, phosphorylation of the β1γ12 dimer on the γ subunit with protein kinase C α regulates its activity in an effector-specific fashion. Because the γ12 subunit is widely expressed, phosphorylation may be an important mechanism for integration of the multiple signals generated by receptor activation.

Differential Sensitivity of P-Rex1 to Isoforms of G Protein βγ Dimers

P-Rex1 is a specific guanine nucleotide exchange factor (GEF) for Rac, which is present in high abundance in brain and hematopoietic cells. P-Rex1 is dually regulated by phosphatidylinositol (3,4,5)-trisphosphate and the Gβγ subunits of heterotrimeric G proteins. We examined which of the multiple G protein α and βγ subunits activate P-Rex1-mediated Rac guanine nucleotide exchange using pure, recombinant proteins reconstituted into synthetic lipid vesicles. AlF–4 activated Gs,Gi,Gq,G12, or G13 α subunits were unable to activate P-Rex1. Gβγ dimers containing Gβ1–4 complexed with γ2 stimulated P-Rex1 activity with EC50 values ranging from 10 to 20 nm. Gβ5γ2 was not able to stimulate P-Rex1 GEF activity. Dimers containing the β1 subunit complexed with a panel of different Gγ subunits varied in their ability to stimulate P-Rex1. The β1γ3, β1γ7, β1γ10, and β1γ13HA dimers all activated P-Rex1 with EC50 values ranging from 20 to 38 nm. Dimers composed of β1γ12 had lower EC50 values (∼112 nm). The farnesylated γ11 subunit is highly expressed in hematopoietic cells; surprisingly, dimers containing this subunit (β1γ11) were also less effective at activating P-Rex1. These findings suggest that the composition of the Gβγ dimer released by receptor activation may differentially activate P-Rex1.

EXPRESSION, PURIFICATION, AND REGULATION OF TWO ISOFORMS OF THE INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE

The level of inositol 1,4,5-trisphosphate in the cytoplasm is tightly regulated by two enzymes, the inositol 1,4,5,5-phosphatase and the inositol 1,4,5-trisphosphate 3-kinase. Two isoforms of the inositol 1,4,5-trisphosphate 3-kinase have been identified, the A form and the B form. The regulatory properties of the two isoforms were compared following overexpression and purification of the proteins from a v-src transformed mammalian cell line. The highly purified, recombinant inositol 1,4,5-trisphosphate 3-kinases were differentially regulated by calcium/calmodulin and via phosphorylation by protein kinase C or the cyclic AMP-dependent protein kinase. Both enzymes had similar affinities for inositol 1,4,5-trisphosphate (K m 2–5 μm). Calcium/calmodulin stimulated the activity of isoform A about 2.5-fold, whereas the activity of isoform B was increased 20-fold. The cyclic AMP-dependent protein kinase phosphorylated the inositol 1,4,5-trisphosphate 3-kinase A to the extent of 0.9 mol/mol and isoform B to 1 mol/mol. Protein kinase C phosphorylated isoform A to the extent of 2 mol/mol and isoform B to 2.7 mol/mol. Phosphorylation of isoform A by the cyclic AMP-dependent protein kinase caused a 2.5-fold increase in its activity when assayed in the absence of calcium/calmodulin, whereas phosphorylation by protein kinase C decreased activity by 72%. The activity of isoform B in the absence of calcium/calmodulin was not affected by phosphorylation using either kinase. When assayed in the presence of calcium/calmodulin, phosphorylation of isoform A by the cyclic AMP-dependent protein kinase increased activity 1.5-fold, whereas phosphorylation of isoform B decreased activity by 45%. Phosphorylation of either isoform A or B by protein kinase C resulted in a 70% reduction of calcium/calmodulin-stimulated activity. Differential expression and regulation of the two inositol 1,4,5-trisphosphate 3-kinase isoforms provides multiple mechanisms for regulating the cytosolic level of inositol 1,4,5-trisphosphate in cells.