Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of G��i-coupled receptors

Chemotaxis is mediated by activation of seven-transmembrane domain, G protein-coupled receptors, but the signal transduction pathways leading to chemotaxis are poorly understood. To identify G proteins that signal the directed migration of cells, we stably transfected a lymphocyte cell line (300-19) with G protein-coupled receptors that couple exclusively to G��q (the m3 muscarinic receptor), G��i (the κ-opioid receptor), and G��s (the β-adrenergic receptor), as well as the human thrombin receptor (PAR-1) and the C-C chemokine receptor 2B. Cells expressing receptors that coupled to G��i, but not to G��q or G��s, migrated in response to a concentration gradient of the appropriate agonist. Overexpression of G�� transducin, which binds to and inactivates free G��γ dimers, completely blocked chemotaxis although having little or no effect on intracellular calcium mobilization or other measures of cell signaling. The identification of G��γ dimers as a crucial intermediate in the chemotaxis signaling pathway provides further evidence that chemotaxis of mammalian cells has important similarities to polarized responses in yeast. We conclude that chemotaxis is dependent on activation of G��i and the release of G��γ dimers, and that G��i-coupled receptors not traditionally associated with chemotaxis can mediate directed migration when they are expressed in hematopoietic cells.

RGS-GAIP, a GTPase-activating Protein for G��i Heterotrimeric G Proteins, Is Located on Clathrin-coated Vesicles

RGS-GAIP (G��-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate G��i/G��q-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein G��i3, which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi- or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion.

GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of G��i���subunits

GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of G-protein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein G��i3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [35S]methionine, two pools of GAIP—a soluble and a membrane-anchored pool—were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membrane-anchored GAIP might be palmitoylated. We found that after labeling with [3H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the G��i subfamily, G��i1, G��i2, G��i3, G��z, and G��o, but not with members of other G�� subfamilies, G��s, G��q, and G��12/13. The C terminus of G��i3 is important for binding because a 10-aa C-terminal truncation and a point mutant of G��i3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of G��i3, which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP’s possible involvement in membrane trafficking.

Platelet signal transduction defect with G�� subunit dysfunction and diminished G��q in a patient with abnormal platelet responses

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5′-[γ-thio]triphosphate (GTP[γS]) was diminished in the patient’s platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of α-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[γS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by G��s) and its inhibition (mediated by G��i) by thrombin in the patient’s platelet membranes were normal. Immunoblot analysis of G�� subunits in the patient’s platelet membranes showed a decrease in G��q (<50%) but not G��i, G��z, G��12, and G��13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein α-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of G��q in thrombin-induced responses.

RGS proteins reconstitute the rapid gating kinetics of G��γ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of G��(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling��� (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein α subunit G��13

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein α subunits were characterized in transfection systems. G��q, G��12, and G��13, but not G��i, activate SRF through RhoA. When G��q, α12, or α13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, G��13, but not G��q or G��12, showed synergistic activation of SRF with GEF115. The synergy between G��13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between G��13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited G��13- and G��12-induced, but not GEF115 itself- or G��q-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of G��12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both G��12 and G��13. Thus, the inhibition of G��12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between G��13 and GEF115 indicates that GEF115 mediates G��13-induced activation of Rho and SRF.

The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families.

Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor.

GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP

We have identified a mammalian protein called GIPC (for GAIP interacting protein, C terminus), which has a central PDZ domain and a C-terminal acyl carrier protein (ACP) domain. The PDZ domain of GIPC specifically interacts with RGS-GAIP, a GTPase-activating protein (GAP) for G��i subunits recently localized on clathrin-coated vesicles. Analysis of deletion mutants indicated that the PDZ domain of GIPC specifically interacts with the C terminus of GAIP (11 amino acids) in the yeast two-hybrid system and glutathione S-transferase (GST)-GIPC pull-down assays, but GIPC does not interact with other members of the RGS (regulators of G protein signaling) family tested. This finding is in keeping with the fact that the C terminus of GAIP is unique and possesses a modified C-terminal PDZ-binding motif (SEA). By immunoblotting of membrane fractions prepared from HeLa cells, we found that there are two pools of GIPC–a soluble or cytosolic pool (70%) and a membrane-associated pool (30%). By immunofluorescence, endogenous and GFP-tagged GIPC show both a diffuse and punctate cytoplasmic distribution in HeLa cells reflecting, respectively, the existence of soluble and membrane-associated pools. By immunoelectron microscopy the membrane pool of GIPC is associated with clusters of vesicles located near the plasma membrane. These data provide direct evidence that the C terminus of a RGS protein is involved in interactions specific for a given RGS protein and implicates GAIP in regulation of additional functions besides its GAP activity. The location of GIPC together with its binding to GAIP suggest that GAIP and GIPC may be components of a G protein-coupled signaling complex involved in the regulation of vesicular trafficking. The presence of an ACP domain suggests a putative function for GIPC in the acylation of vesicle-bound proteins.

The problem of the spreading of a liquid film along a solid surface: A new mathematical formulation

A new mathematical model is proposed for the spreading of a liquid film on a solid surface. The model is based on the standard lubrication approximation for gently sloping films (with the no-slip condition for the fluid at the solid surface) in the major part of the film where it is not too thin. In the remaining and relatively small regions near the contact lines it is assumed that the so-called autonomy principle holds—i.e., given the material components, the external conditions, and the velocity of the contact lines along the surface, the behavior of the fluid is identical for all films. The resulting mathematical model is formulated as a free boundary problem for the classical fourth-order equation for the film thickness. A class of self-similar solutions to this free boundary problem is considered.

The influence of the flow of the reacting gas on the conditions for a thermal explosion

The classical problem of thermal explosion is modified so that the chemically active gas is not at rest but is flowing in a long cylindrical pipe. Up to a certain section the heat-conducting walls of the pipe are held at low temperature so that the reaction rate is small and there is no heat release; at that section the ambient temperature is increased and an exothermic reaction begins. The question is whether a slow reaction regime will be established or a thermal explosion will occur. The mathematical formulation of the problem is presented. It is shown that when the pipe radius is larger than a critical value, the solution of the new problem exists only up to a certain distance along the axis. The critical radius is determined by conditions in a problem with a uniform axial temperature. The loss of existence is interpreted as a thermal explosion; the critical distance is the safe reactor’s length. Both laminar and developed turbulent flow regimes are considered. In a computational experiment the loss of the existence appears as a divergence of a numerical procedure; numerical calculations reveal asymptotic scaling laws with simple powers for the critical distance.

The thermal explosion revisited

The classical problem of the thermal explosion in a long cylindrical vessel is modified so that only a fraction α of its wall is ideally thermally conducting while the remaining fraction 1−α is thermally isolated. Partial isolation of the wall naturally reduces the critical radius of the vessel. Most interesting is the case when the structure of the boundary is a periodic one, so that the alternating conductive α and isolated 1−α parts of the boundary occupy together the segments 2π/N (N is the number of segments) of the boundary. A numerical investigation is performed. It is shown that at small α and large N, the critical radius obeys a scaling law with the coefficients depending on N. For large N, the result is obtained that in the central core of the vessel the temperature distribution is axisymmetric. In the boundary layer near the wall having the thickness ≈2πr0/N (r0 is the radius of the vessel), the temperature distribution varies sharply in the peripheral direction. The temperature distribution in the axisymmetric core at the critical value of the vessel radius is subcritical.

Very intense pulse in the groundwater flow in fissurized-porous stratum

An asymptotic solution is obtained corresponding to a very intense pulse: a sudden strong increase and fast subsequent decrease of the water level at the boundary of semi-infinite fissurized-porous stratum. This flow is of practical interest: it gives a model of a groundwater flow after a high water period or after a failure of a dam around a collector of liquid waste. It is demonstrated that the fissures have a dramatic influence on the groundwater flow, increasing the penetration depth and speed of fluid penetration into the stratum. A characteristic property of the flow in fissurized-porous stratum is the rapid breakthrough of the fluid at the first stage deeply into the stratum via a system of cracks, feeding of porous blocks by the fluid in cracks, and at a later stage feeding of advancing fluid flow in fissures by the fluid, accumulated in porous blocks.

Corticotropin-releasing hormone causes antidiuresis and antinatriuresis by stimulating vasopressin and inhibiting atrial natriuretic peptide release in male rats

In both normally hydrated and volume-expanded rats, there was a biphasic effect of corticotropin-releasing hormone (CRH) (1–10 μg, i.v.) on renal function. Within the first hour, CRH caused antidiuresis, antinatriuresis, and antikaliuresis together with reduction in urinary cGMP output that, in the fourth hour, were replaced by diuresis, natriuresis, and kaliuresis accompanied by increased cGMP output. Plasma arginine vasopressin (AVP) concentrations increased significantly within 5 min, reached a peak at 15 min, and declined by 30 min to still-elevated values maintained for 180 min. Changes in plasma atrial natriuretic peptide (ANP) were the mirror image of those of AVP. Plasma ANP levels were correlated with decreased ANP in the left ventricle at 30 min and increased ANP mRNA in the right atrium at 180 min. All urinary changes were reversed by a potent AVP type 2 receptor (V2R) antagonist. Control 0.9% NaCl injections evoked an immediate increase in blood pressure and heart rate measured by telemetry within 3–5 min. This elevation of blood pressure was markedly inhibited by CRH (5 μg). We hypothesize that the effects are mediated by rapid, direct vasodilation induced by CRH that decreases baroreceptor input to the brain stem, leading to a rapid release of AVP that induces the antidiuresis by direct action on the V2Rs in the kidney. Simultaneously, acting on V2Rs in the heart, AVP inhibits ANP release and synthesis, resulting in a decrease in renal cGMP output that is responsible for the antinatriuretic and antikaliuretic effects.

Self-similar intermediate asymptotics for a degenerate parabolic filtration-absorption equation

The equation ∂tu = u∂xx2u − (c − 1)(∂xu)2 is known in literature as a qualitative mathematical model of some biological phenomena. Here this equation is derived as a model of the groundwater flow in a water-absorbing fissurized porous rock; therefore, we refer to this equation as a filtration-absorption equation. A family of self-similar solutions to this equation is constructed. Numerical investigation of the evolution of non-self-similar solutions to the Cauchy problems having compactly supported initial conditions is performed. Numerical experiments indicate that the self-similar solutions obtained represent intermediate asymptotics of a wider class of solutions when the influence of details of the initial conditions disappears but the solution is still far from the ultimate state: identical zero. An open problem caused by the nonuniqueness of the solution of the Cauchy problem is discussed.

The miniaturized nuclear genome of eukaryotic endosymbiont contains genes that overlap, genes that are cotranscribed, and the smallest known spliceosomal introns.

Chlorarachniophyte algae contain a complex, multi-membraned chloroplast derived from the endosymbiosis of a eukaryotic alga. The vestigial nucleus of the endosymbiont, called the nucleomorph, contains only three small linear chromosomes with a haploid genome size of 380 kb and is the smallest known eukaryotic genome. Nucleotide sequence data from a subtelomeric fragment of chromosome III were analyzed as a preliminary investigation of the coding capacity of this vestigial genome. Several housekeeping genes including U6 small nuclear RNA (snRNA), ribosomal proteins S4 and S13, a core protein of the spliceosome [small nuclear ribonucleoprotein (snRNP) E], and a cip-like protease (clpP) were identified. Expression of these genes was confirmed by combinations of Northern blot analysis, in situ hybridization, immunocytochemistry, and cDNA analysis. The protein-encoding genes are typically eukaryotic in overall structure and their messenger RNAs are polyadenylylated. A novel feature is the abundance of 18-, 19-, or 20-nucleotide introns; the smallest spliceosomal introns known. Two of the genes, U6 and S13, overlap while another two genes, snRNP E and clpP, are cotranscribed in a single mRNA. The overall gene organization is extraordinarily compact, making the nucleomorph a unique model for eukaryotic genomics.