J Biol Chem, Vol. 274, Issue 38, 26860-26868, September 17, 1999

RGS1 Is Expressed in Monocytes and Acts as a GTPase-activating Protein for G-protein-coupled Chemoattractant Receptors^*

Bernd Denecke, Anke Meyerdierks, and Erik C. Böttger

From the Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, D-30625 Hannover, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The leukocyte response to chemoattractants is transduced by the interaction of transmembrane receptors with GTP-binding regulatory proteins (G-proteins). RGS1 is a member of a protein family constituting a newly appreciated and large group of proteins that act as deactivators of G-protein signaling pathways by accelerating the GTPase activity of G-protein  subunits. We demonstrate here that RGS1 is expressed in human monocytes; by immunofluorescence and subcellular fractionation RGS1 was localized to the plasma membrane. By using a mixture of RGS1 and plasma membranes, we were able to demonstrate GAP activity of RGS1 on receptor-activated G-proteins; RGS1 did not affect ligand-stimulated GDP-GTP exchange. We found that RGS1 desensitizes a variety of chemotactic receptors including receptors for N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and C5a. Interaction of RGS proteins and ligand-induced G-protein signaling can be demonstrated by determining GTPase activity using purified RGS proteins and plasma membranes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The accumulation of neutrophils and mononuclear phagocytes is a major characteristic of inflammatory processes. Many of the responses of monocytes to infection are regulated by chemoattractant or proinflammatory factors. N-Formyl-methionyl peptides (e.g. N-formyl-methionyl-leucyl-phenylalanine (fMLP)),¹ complement component C5a, and leukotriene B4 (LTB4) are some of the most potent chemoattractant stimuli (1-5). These small peptide or lipid factors bind to high affinity cell surface receptors and trigger chemotactic migration and activation of the inflammatory response (6-8). G-proteins, or heterotrimeric GTP-binding regulatory proteins, have been implicated in regulation of the response to chemoattractant ligands through biochemical studies of the effects of GTP analogues on chemoattractant factor binding and activation of phospholipases in permeabilized cells and membranes (3). In addition, pertussis toxin, which blocks the activation of certain G-proteins, inhibits the chemoattractant response (4).

Heterotrimeric G-proteins are essential elements of many transmembrane signaling pathways involved in coupling receptors for extracellular mediators to a variety of second messenger-generated effector enzymes or ion channels (9, 10). Heterotrimeric G-proteins are composed of , , and  subunits (8, 11). The inactive G-protein is a GDP-bound heterotrimer. Ligand-bound activated receptors catalyze the exchange of GDP by GTP, leading to dissociation of the  subunit from the dimer and signal transduction by the separated G-protein subunits (G and G). Recent experiments provided evidence that chemotaxis is mediated by G dimers released by activation of G-coupled receptors (12, 13). GTP hydrolysis by the intrinsic GTPase activity of the G subunit deactivates G-proteins by allowing heterotrimers to reform (14).

Many G-protein-mediated signaling pathways become desensitized after stimulation. One such mechanism involves phosphorylation of the G-protein-coupled receptor by protein kinases thereby uncoupling the receptor from interaction with the transducing G-protein (15, 16). A recently discovered mechanism of desensitization results from interaction of the G protein with members of a family of proteins termed RGS proteins (regulators of G-protein signaling). At least 18 members of this protein family have been described in mammals (17-20). Members of the mammalian RGS family have been shown to induce desensitization in yeast (21, 22), suggesting desensitization functions for them in mammals as well (17). The molecular mechanism of RGS-induced desensitization was revealed by the demonstration that RGS proteins in vitro interact specifically with the activated form of G subunits and accelerate hydrolysis of GTP (19, 23-26).

Studies of the phototransduction cascade have demonstrated that different modes of desensitization complement each other. Phosphorylation of the receptor (rhodopsin) leads to rapid deactivation controlling the amplitude of the response, whereas G-protein (transducin) deactivation by hydrolysis of GTP is rate-limiting for shut off of the phototransduction cascade and controls recovery (27).

Patterns of specificity between individual RGS proteins and subfamilies of G proteins are only beginning to emerge (28). RGS proteins investigated so far can serve as GTPase-activating proteins (GAPs) for the G_i and G_q but not the G_s subfamily of  subunits. In the experiments purified, GTP-loaded G subunits were used as substrates under conditions in which one round of hydrolysis could be initiated by the prebound nucleotide (19, 23-26). Although these experiments have established the GAP activity of RGS proteins, it has not been possible to define the specific receptor to which a particular RGS protein couples. Besides acting as a GAP, there is evidence that RGS proteins may also interfere directly with activated G subunits, e.g. by occlusion of the binding site of G for phospholipase C (28, 29). Little information is available on the distribution and the subcellular localization of RGS proteins, with the exception of GAIP, which was demonstrated to be membrane-anchored (20).

In this paper we investigate the distribution and subcellular localization of RGS1. By showing that RGS1 enhances chemoattractant-induced GTPase activity in plasma membranes from Bt₂cAMP-treated human monocytic U937 and THP1 cells, we provide evidence that the receptors for the chemoattractants fMLP, C5a, and LTB4 are desensitized by RGS1 and that a mixture of ligand-stimulated plasma membranes and recombinant RGS proteins can be used to investigate G-protein and RGS protein interaction. The use of ligand-stimulated plasma membranes allowed us to rule out a possible activity of RGS1 as promoter of GDP-GTP exchange.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals and Antibodies-- The following reagents were obtained as indicated: [-³²P]CTP, [-³²P]GTP, [-³²P]GTP, [-³⁵S]GTPS (Amersham Pharmacia Biotech); dibutyryl-cAMP (Bt₂cAMP), C5a, N-formyl-Met-Leu-Phe (fMLP), 12-tetradecanoyl-phorbol-13-acetate (TPA) (Sigma); leukotriene B4 (LTB4), pertussis toxin (Calbiochem); IFN (Imukin®-Thomae); mouse monoclonal antibody (12CA5) reactive with the HA tag, anti-digoxigenin-AP Fab fragment (Roche Molecular Biochemicals); mouse monoclonal antibody (T56/14) reactive with the transferrin receptor/CD71 (Oncogene Science); goat anti-mouse fluorescein isothiocyanate, goat anti-mouse peroxidase, goat anti-rabbit peroxidase (Jackson ImmunoResearch); Alexa^TM 488 goat anti-rabbit (Molecular Probes). Polyclonal antisera against RGS1 and WRS/tryptophanyl-tRNA synthetase were made in rabbits by standard immunization protocols using a GST-RGS1 fusion protein and a MBP-WRS fusion protein, respectively.

Polyclonal RGS1 antiserum was affinity purified using N-hydroxysuccinimide-activated Sepharose High Performance HiTrap® columns (Amersham Pharmacia Biotech) covalently coupled with (i) purified GST protein, (ii) purified MBP protein, and (iii) purified MBP-RGS1 fusion protein, respectively, according to the manufacturer's instructions. Antibody activity of the fractions was tested by Western blot analysis.

Cloning Procedures

cDNA Libraries and Isolation of RGS1 cDNA-- Pooled poly(A)⁺ RNA isolated from untreated U937 and THP1 cells was used for construction of a directional cDNA library in EcoRI-XbaI-digested  GEM®2 vector arms according to the manufacturer's instructions (Promega); subsequently biotinylated cRNA was generated utilizing the "MEGAscript^TM T7 in Vitro Transcription kit" (Ambion) modified by exchanging UTP for Biotin-21-UTP (CLONTECH).

Poly(A)⁺ RNA from TPA/IFN- and IFN-treated U937 and THP1 cells was pooled (induced poly(A)⁺ RNA pool), and cDNA was generated using the ZAP-cDNA® synthesis kit (Stratagene). After alkaline hydrolysis to remove RNA templates, the induced cDNA was subtracted twice with uninduced cRNA. Biotinylated sequences and hybrids were removed by adding 20 µg of streptavidin (Life Technologies, Inc.) and extraction with phenol. Second strand synthesis was performed after rehybridization of the subtracted single strand cDNA to the induced poly(A)⁺ RNA. cDNA was unidirectionally ligated into  ZAP® II vector digested with EcoRI-XhoI (Stratagene) and in vitro packaged using ZAP-cDNA® Gigapack® III (Stratagene).

The subtracted library was screened for TPA/IFN-induced genes using a highly labeled [³²P]-cDNA probe, which was prepared from the induced poly(A)⁺ RNA pool and subtracted twice with cRNA from uninduced cells using the above described procedure. By screening 75,000 colonies from the subtracted library with the subtracted cDNA probe 2789 single clones were isolated. Five of the cDNAs analyzed were coding for RGS1 (30). Excision of the pBluescript phagemid from the  ZAP® II vector was performed using the ExAssist^TM helper phage resulting in RGS1/pBluescript vector SK().

Recombinant RGS1 Constructs-- The RGS1 cDNA coding for 196 aa was amplified by PCR using oligonucleotide 5'-CGGAATTCCCAGGAATGTTCTTCTCTG-3' (aa position +2) and oligonucleotide 5'-GCTCTAGACTGGCACATTCCTTCGTG-3' (63 nucleotides 3' of the stop codon). The PCR product was cloned into pT7-vector (Novagen), digested with BglII and SalI, and ligated in frame into the BamHI/SalI sites of pGEX-4T-1 (Amersham Pharmacia Biotech) resulting in plasmid pGEX4T1 RGS1. The DNA sequence was determined by automated DNA sequencing (Applied Biosystems and Perkin-Elmer) to confirm its proper insertion into the vector. The recombinant plasmid was transformed into Escherichia coli DH5 and induced at an A₆₀₀ of 0.5 by addition of isopropyl-1-thio--D-galactopyranoside (0.3 mM final concentration) to express the GST-RGS1 fusion protein. After a 2-h induction at 37 °C, cells were harvested, and GST-RGS1 fusion protein was prepared and purified in a batch procedure using glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

RGS1 cDNA was amplified by PCR using oligonucleotide 5'-CGGGATCCCCAGGAATGTTCTTCTCTG-3' to introduce a BamHI at the 5'-end of RGS1 (aa position +2), and oligonucleotide 5'-GCGGATCCCTTTAGGCTATTAGCCTGC-3' to introduce a BamHI site at the 3'-end of RGS1 (aa position 195). The 601-base pair PCR product was digested with BamHI and subcloned into the BamHI site of the mammalian expression vector pEVRFO (31), resulting in expression of carboxyl-terminal HA-tagged RGS1, termed RGS1 HA tag.

The same BamHI-digested 601-base pair PCR product was subcloned into the BamHI site of His tag vector pQE30 (Qiagen). The recombinant plasmid was transformed into E. coli BL21 (DE3), and expression of the fusion protein was induced with 1 mM isopropyl-1-thio--D-galactopyranoside. After a 2-h induction at 37 °C, cells were harvested, and the His-tagged RGS1 fusion protein was purified by affinity chromatography using a nickel-nitrilotriacetic acid affinity column (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Cell Culture and Transfection-- Jurkat, Raji, THP1, HL-60, and U937 cells were grown in suspension in RPMI 1640 medium (Life Technologies, Inc.); HaCat and HeLa S3 cells were grown as adherent cells using Dulbecco's modified Eagle's medium; CHO cells were grown in Dulbecco's modified Eagle's medium/HAM's F12 medium (Life Technologies, Inc.). For treatment with 12-tetradecanoyl-phorbol-13-acetate (TPA), cells were seeded at densities of 5 × 10⁵/ml. TPA was added for 48 h at a final concentration of 20 nM for THP1 cells, and at a final concentration of 10 nM for U937, Jurkat, Raji, HaCat, and HeLa S3 cells. Recombinant human IFN was used in a final concentration of 1000 units/ml for 24 h. For induction with Bt₂cAMP the medium was supplemented with 1 mM Bt₂cAMP for 72 h.

For immunofluorescence studies CHO cells (5 × 10⁴/well) were plated on 24-well tissue culture plates covered with clear glass coverslips (12 mm diameter, Assistent, Sondheim/Rhön, Germany). Cells were cultured overnight and transfected with LipofectAMINE^TM reagent (Life Technologies, Inc.) according to the manufacturer's instructions.

For preparing cell extracts CHO cells were seeded at a density of 5 × 10⁵/6-cm dish, cultured overnight, and transfected with LipofectAMINE^TM reagent (Life Technologies, Inc.) according to the manufacturer's instructions.

Preparation of Human Peripheral Blood Leukocytes-- PBMCs were isolated from buffy coats by Ficoll-Paque gradient centrifugation. B- and T-cells were isolated from the mononuclear fraction using MACS Beads (Miltenyi) (anti-CD19 for B-cells and anti CD3 for T-cells). Monocytes were prepared by adherence. Fluorescence-activated cell sorter analysis revealed greater than 95% purity of monocytes, T-cells, and B-cells.

RNA Analysis-- Total RNA was prepared using the guanidinium isothiocyanate/cesium chloride gradient procedure (32, 33). 15 µg of total RNA/slot were separated by electrophoresis through denaturing 1.4% agarose-formaldehyde gels and transferred by capillary blotting with 20× SSC onto nylon membranes (Hybond-N, Amersham Pharmacia Biotech) according to the manufacturer's recommendations. The RNA was covalently UV cross-linked to the membrane (Stratagene UV cross-linker).

A digoxigenin-labeled RGS1 riboprobe was generated in a standard transcription assay with T3 RNA polymerase using the digoxigenin-RNA labeling kit (Roche Molecular Biochemicals) after linearization of the RGS1/pBluescript vector SK() (Stratagene) with XhoI.

Following hybridization the membranes were developed by DIG detection according to the manufacturer's luminescent detection protocol (CSPD, Roche Molecular Biochemicals).

Western Blot Analysis-- Cells were pelleted by centrifugation and washed with PBS. Aliquots of 1 × 10⁶ cell pellets were lysed in SDS-PAGE sample buffer, separated by SDS-disc gel electrophoresis, and transferred to nitrocellulose membrane by electroblotting. After blocking the membranes with blocking buffer (PBS containing 1.5% nonfat milk and 0.05% Tween 20) for 1 h at RT, membranes were washed one time with WP (PBS containing 0.1% Tween 20 and 0.1% Nonidet P-40) followed by incubation for 2 h at RT with anti-RGS1 rabbit antiserum and mAb 12CA5, respectively. Membranes were washed four times with WP and incubated with peroxidase-conjugated goat anti-rabbit and goat anti-mouse, respectively, for 1 h at RT. After washing four times with WP, relevant proteins were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).

Preparation of Plasma Membranes-- Cells were washed twice with PBS, and the pellet of 2 × 10⁷ cells was stored at 70 °C prior to membrane production. The frozen cell pellet was thawed by resuspending in 5 volumes of ice-cold TEmp buffer (10 mM Tris/HCl, 0.1 mM EDTA, 4 µg/ml leupeptin, 28 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, pH 7.5). Subsequently, cells were homogenized by 25 firm strokes using a Dounce homogenizer (5 ml) with a tight fitting pestle and passed through a 27-gauge syringe needle. The homogenate was centrifuged at 500 × g for 15 min to pellet unbroken cells and nuclei. Membranes were collected by centrifugation of the supernatant at 100,000 × g for 45 min at 4 °C. The supernatant was collected as cytosolic fraction. The membrane containing pellet was washed in TEmp buffer and recentrifuged at 100,000 × g for 45 min at 4 °C. The pellet was then resuspended in TEmp buffer at a final protein concentration between 1 and 3 mg/ml, aliquoted, and stored until use at 70 °C. Protein content was determined by the Bradford method using bovine serum albumin as standard.

Measurement of GTP Hydrolysis-- Hydrolysis of [-³²P]GTP was determined in a reaction mixture (100 µl) containing 3 µg of membrane protein, 1 mM App(NH)P, 1 mM ATP, 1 mM ouabain, 10 mM creatine phosphate, 2.5 units/ml creatine phosphokinase, 100 mM NaCl, 5 mM MgCl₂, 2 mM dithiothreitol, 0.1 mM EDTA, 10 mM Tris/HCl, pH 7.4, 0.5 µM GTP, and [-³²P]GTP (0.5 µCi/reaction mixture). Agonists were added as indicated in the figures and reactions started by addition of membranes to the incubation mixture and incubation at 37 °C. Reactions were terminated by adding 900 µl of a 5% (w/v) ice-cold activated charcoal slurry in 20 mM phosphoric acid, pH 2.3, resulting in a total volume of 1000 µl. To pellet the charcoal along with unhydrolyzed [-³²P]GTP, tubes were centrifuged at 12,000 × g for 30 min at 4 °C. 500 µl of the supernatant fluid containing the free [³²P_i] were removed and used for determination of Cerenkov radiation.

Low affinity hydrolysis of [-³²P]GTP was assessed by incubating in the presence of 100 µM GTP. Blank values were determined by replacement of membrane protein with TE (10 mM Tris/HCl, 1 mM EDTA, pH 8.0); the maximal GTP hydrolysis was determined by counting 500 µl of a 1:10 diluted mixture directly. Low affinity hydrolysis and blank values were less than 1% of the total [-³²P]GTP present in the assay.

After subtracting the blank values, the rate of hydrolysis of GTP was calculated as followed: C/S.A. × 2 × 1000/P × 1/t; where C indicates the counts determined in the 500-µl sample; S.A. indicates the specific activity of the GTP (each assay tube contained 50 pmol of cold GTP; the specific activity per pmol is the maximal activity/50); P indicates the amount of protein present in µg; and t indicates the time of the assay. This calculation gives the rate of hydrolysis of GTP in pmol/min/mg of membrane protein.

Measurement of GTP Binding-- Binding assays were performed at 30 °C in an incubation mixture (100 µl) containing 5 µg of membrane protein, 10 mM Tris/HCl, pH 7.4, 5 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol, 150 mM NaCl, 1 µM GDP, and 112.5 fmol of [³⁵S]GTPS. Agonists were added as noted in the figures, and reactions were started by the addition of membranes to the prewarmed (30 °C) reaction mixture. The reaction was terminated at various time points by rapid filtration through Whatman GF/C glass-fiber filters under vacuum and three washes with 5 ml of 10 mM Tris/HCl, 0.1 mM EDTA, pH 7.5. Radioactivity was determined by liquid scintillation counting (dried GF/C filters were placed in a scintillation vial along with 20 ml of scintillation fluid (LSC safety mixture, Baker/Holland), mixed, and left in the dark for 12 h). Nonspecific binding is defined by the binding that was not competed for by 10 µM unlabeled GTPS; nonspecific binding was less than 1% of the total [³⁵S]GTPS present in the assay. Blank values were determined by replacing membrane protein with TE.

Immunofluorescence and Confocal Microscopy-- CHO cells cultured on coverslips were fixed in PBS, 3% paraformaldehyde for 10 min at room temperature (RT) and permeabilized for 15 min with 0.05% Nonidet P-40. TPA-treated THP1 cells were trypsinated with trypsin-EDTA (Life Technologies, Inc.), washed with PBS, placed on poly-L-lysine-coated glass microscope slides for 15 min, fixed in 50% methanol, 50% acetone for 10 min at 4 °C, and permeabilized for 15 min with 0.1% Saponin. Following permeabilization cells were rinsed three times with PBS, incubated with blocking buffer (PBS containing 0, 1% Tween 20 and 10% fetal calf serum) for 30 min at RT, followed by incubation with the first antibody diluted in blocking buffer for 2 h at RT. After four washes with PBS, cells were incubated with the secondary antibody diluted in blocking buffer for 1 h at RT. Cells were washed four times with PBS and mounted with coverslips in PermaFluor (Immunotech) to stabilize fluorescence.

Laser scanning microscopy was performed by M. Rohde (GBF, Braunschweig, Germany) using a Zeiss inverted microscope and a scanning confocal imaging system (MRC1024-UV, Bio-Rad) equipped with a 5-milliwatt krypton/argon laser (488, 568, and 647 nm) and a T1/E2 filter set for detection of fluorescein isothiocyanate-labeled antibodies. Images were collected with a PlanNeofluar 63× oil NA 1.25 objective and were processed using Bio-Rad LaserSharp 3.1T software. Optical sections were obtained at 0.2-µm intervals (CHO cells) and 1.0-µm intervals, respectively (THP1 cells).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RGS1 Is Expressed in Cells of the Hematopoietic Lineage-- Human monocyte THP1 and U937 cells were differentiated by treatment with TPA and IFN. Genes expressed during differentiation were isolated by subtractive cloning procedures. One cDNA obtained was identified as RGS1. Northern blot analysis demonstrated that expression of RGS1 in THP1 and U937 cells was restricted to treatment with TPA (Fig. 1A). In line with the original characterization of RGS1 as a protein inducible by mitogenic stimuli in B-cells (30, 34), significant levels of RGS1 mRNA expression were observed in TPA-treated Raji B-cells (Fig. 1B); no expression was found in Jurkat T-cells, epithelial HeLa cells, and HaCat keratinocytes with or without prior TPA treatment (data not shown). Analysis of purified populations of monocytes, B-cells, and T-cells from peripheral blood demonstrated that expression of RGS1 mRNA is confined to monocytes (Fig. 1B).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   RGS1 mRNA is expressed in U937, THP1, and Raji cells after TPA treatment; RGS1 mRNA is constitutively expressed in peripheral blood monocytes; and RGS1 mRNA is not expressed in peripheral blood B-cells and T-cells. A, untreated U937 and THP1 cells (lanes 1 and 5); U937 and THP1 cells treated with TPA (lanes 2 and 6); U937 and THP1 cells treated with IFN (lanes 3 and 7); U937 and THP1 cells treated with TPA/IFN (lanes 4 and 8). B, total RNA from untreated THP1 and Raji cells (lanes 1 and 7); THP1 and Raji cells treated with TPA (lanes 2 and 8); total RNA from purified populations of B-cells (lane 3), T-cells (lane 4) and monocytes (lane 5). Lane 6 is total RNA from peripheral blood monocytes treated with TPA. 15 µg of total RNA from the indicated cell lines and PBMCs, respectively, were analyzed; equal loading and integrity of the RNA samples was confirmed by ethidium bromide staining of the rRNA bands.

Polyclonal antibodies were raised against recombinant RGS1 protein. Specificity of the antibody was controlled by analysis of CHO cells transfected with an HA-tagged RGS1 expression construct (Fig. 2A) and demonstrated a major protein of 25 kDa. Due to the insertion of the HA tag, the recombinant protein is slightly larger than the native RGS1 protein; the mouse monoclonal antibody 12CA5 directed against the HA epitope recognized a protein of identical molecular weight in protein extracts from transfected CHO cells (lower part of Fig. 2A).

View larger version (46K): [in this window] [in a new window]   Fig. 2.   Western blot analysis of RGS1 expression using RGS1 antiserum. A, purified GST-RGS1 fusion protein and cell extracts of CHO cells (1 × 106 cells) transfected with RGS1 HA tag. Proteins were separated by 15% SDS-disc gel electrophoresis. GST-RGS1 (45 kDa) and RGS1-HA (25 kDa) were detected by RGS1 antiserum (upper part); the blot was stripped and reprobed with anti-HA mAb 12CA5 (lower part). B, affinity purification of RGS1 antiserum. Whole cell extract of TPA-treated THP1 cells was incubated with RGS1 antiserum (lane 1), affinity purified RGS1 antiserum (lane 2), and affinity purified RGS1 antiserum preincubated with GST-RGS1 fusion protein (lane 3). C, RGS1 is expressed in monocytes. Whole cell extracts of 1 × 106 cells from the indicated PBMCs were analyzed: purified populations of monocytes (lane 2), B-cells (B, lane 3), and T-cells (T, lane 4). Con, control. Lane 1 is a control for RGS1 expression (whole cell extract from 1 × 106 TPA treated THP1 cells). D, RGS1 protein expression is induced by TPA treatment in U937, THP1, and Raji cells. Whole cell extracts (1 × 106 cells) from the indicated cell lines were analyzed: untreated THP1, U937, HeLa, HaCat, Raji, and Jurkat cells (lane 1); THP1, U937, HeLa, HaCat, Raji, and Jurkat cells treated with TPA (lane 2).

In Western blots of total proteins from TPA-treated THP1 cells, the RGS1 antiserum recognized a protein of 22 kDa (Fig. 2B, lane 1), which corresponds to the predicted molecular weight of RGS1. To eliminate cross-reactivity, the antiserum was purified by affinity chromatography (compare Fig. 2B, lane 2 with lane 1). Specificity of the antibody was further demonstrated by preincubation with excess RGS1 antigen eliminating the recognition of the 22-kDa protein (Fig. 2B, lane 3).

Western blots of total cellular proteins confirmed the results of the mRNA analysis. RGS1 expression in peripheral blood mononuclear cells was restricted to monocytes (Fig. 2C). RGS1 expression in Raji B-cells and in cells of the monocyte lineage (U937 and THP1) was observed only after treatment with TPA (Fig. 2D). For unknown reasons RGS1 frequently appears as a double band in Western blot analysis.

RGS1 Localizes to the Cell Membrane-- The distribution of RGS1 was assessed in crude membrane (100,000 × g pellet) and cytosolic (100,000 × g supernatant) fractions prepared from CHO cells transiently expressing HA-tagged RGS1. RGS1 was associated with the membrane fraction (Fig. 3A, compare lanes 2 and 3). When this fraction was treated with 0.1 M Na₂CO₃ (pH 11.3) to strip peripheral membrane proteins (35), RGS1 to a large extent remained associated with the membrane fraction (see Fig. 3A, lanes 4 and 5), indicating that RGS1 in part behaves as an integral membrane protein. Digestion of the membrane pellet with proteinase K resulted in complete destruction of RGS1 (Fig. 3A, lanes 6 and 8) suggesting that RGS1 faces the cytoplasm rather than the lumen of an organelle.

View larger version (55K): [in this window] [in a new window]   Fig. 3.   RGS1 is a membrane-associated protein. A, resistance to stripping with Na2CO3 and susceptibility to digestion with proteinase K (Prot. K) indicates that RGS1 is a membrane-anchored protein and faces the cytoplasm. Postnuclear supernatant (lane 1) from CHO cells transiently expressing RGS1-HA was centrifuged at 100,000 × g for 1 h at 4 °C to yield a crude membrane fraction (lane 2) and a cytosolic fraction (cytopl., lane 3). 75 µg of crude membrane fraction were treated with the following: 1) 0.1 M Na2CO3, pH 11.3; 2) PBS; or 3) 50 µg/ml proteinase K for 30 min at 4 °C and centrifuged at 100,000 × g for 1 h at 4 °C. Proteins, i.e. supernatants (s.n.) and pellets (p.), were separated by a 15% SDS-disc gel electrophoresis, immunoblotted with 12CA5 mAb (anti-HA), and detected by ECL. Most of the RGS1-HA is associated with the pellet fraction of cellular extracts (compare lane 2 with lane 3). RGS1 remains in part associated with the membrane fraction after Na2CO3 treatment (compare lane 4 with lane 5); as a control treatment with PBS retained RGS1 in the membrane fraction (compare lanes 6 and 7). Proteinase K treatment results in complete digestion of RGS1 which is detectable neither in the membrane fraction (lane 8) nor in the soluble fraction (lane 9). B, confocal laser scanning microscopy demonstrates that RGS1 localizes to the cell membrane. CHO cells were transiently transfected with expression plasmid RGS1 HA tag. Localization of the HA-tagged RGS1 was performed as described under "Experimental Procedures" using the 12CA5 antibody. Optical sections obtained at 0.2-µm intervals are shown. C, endogenous RGS1 localizes to the membrane fraction. Postnuclear supernatant from TPA-treated THP1 cells was centrifuged at 100,000 × g for 1 h at 4 °C to yield a crude membrane fraction (lane 1) and a cytosolic fraction (lane 2). Proteins were separated by a 15% SDS-disc gel electrophoresis, immunoblotted with affinity purified RGS1 antiserum (upper part), WRS/tryptophanyl-tRNA synthetase antiserum (middle part), and AB-1 mAb directed against transferrin receptor/CD71 (TR, lower part) and detected by ECL. Most of the RGS1 is associated with the membrane fraction of cellular extracts (compare lane 1 with lane 2). WRS/tryptophanyl-tRNA synthetase is mainly associated with the cytosolic fraction; the transferrin receptor is found in the membrane fraction. D, confocal laser scanning microscopy demonstrates that endogenous RGS1 in TPA-treated THP1 cells localizes to the cell membrane. Localization of RGS1 was performed as described under "Experimental Procedures" using affinity purified RGS1 antiserum. Optical sections obtained at 1.0-µm intervals are shown (A-O); a light micrograph of the same cell is given in P.

Confocal laser scan microscopy and immunofluorescence was used to analyze the subcellular expression of RGS1 in transfected CHO cells. Staining with anti-HA antibody 12CA5 was predominantly observed at the cytoplasmic membrane (Fig. 3B).

The subcellular distribution of endogenous RGS1 was investigated in TPA-treated THP1 cells using affinity purified antiserum (Fig. 3C, upper part). To control for the cell fractionation procedure (Fig. 3C, lane 1, membrane fraction; lane 2, cytosolic fraction), the fractions were incubated with antiserum directed against the cytosolic localized protein WRS/tryptophanyl-tRNA synthetase (36) (Fig. 3C, middle part), and a monoclonal antibody directed against the membrane-bound transferrin receptor (37) (Fig. 3C, lower part). Endogenous RGS1 was found to be mainly associated with the membrane fraction; as expected WRS is found in the cytosolic fraction, and the transferrin receptor localizes to the membrane fraction.

Immunofluorescence of TPA-treated THP1 cells using confocal laser scan microscopy demonstrated that membrane-bound RGS1 localizes to the cytoplasmic membrane (Fig. 3D).

Demonstration of RGS1 GAP Activity upon fMLP-stimulated GTP Hydrolysis, RGS1 Accelerates GTP Hydrolysis Mediated by the Chemoattractant fMLP-- Treatment with the membrane-permeable cAMP analogue Bt₂cAMP increases expression of chemoattractant receptors in cells of the monocytic lineage. Fig. 4 shows that fMLP-stimulated GTP hydrolysis was readily detectable in cell membrane preparations from Bt₂cAMP-treated U937 cells; the accumulation of hydrolyzed GTP was linear for up to 30 min under the assay conditions used.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Kinetics of GTP hydrolysis after addition of fMLP, RGS1, or fMLP/RGS1. GTPase activity was determined in plasma membranes of Bt2cAMP-treated U937 cells (3 µg). fMLP and GST-RGS1 were used at concentrations of 1 µM. The basal activity (no addition) was subtracted from the stimulated GTPase activity. The data shown are means ± S.D. of three individual experiments, each consisting of duplicate determinations.

In assays that assess steady-state hydrolysis of GTP, the rate of catalysis may be limited by GDP dissociation. Accordingly, GAP activity of RGS1 has been investigated previously using recombinant G_i proteins, as these G proteins can be prepared in their GTP-bound form by incubation with the nucleotide triphosphate in the absence of Mg²⁺, and the GTP-G substrates can then be used in single turnover assays.

To study the effect of RGS1 on the receptor-stimulated GTPase cycle, we used cell membranes from U937 cells treated with Bt₂cAMP. RGS1 markedly accelerated fMLP receptor-stimulated GTP hydrolysis over the complete period of the assay (Fig. 4). This effect was dependent on the presence of the ligand fMLP, demonstrating that RGS1 acts as an GTPase-activating protein (GAP). Under the conditions used the rate of GTP hydrolysis was apparently not limited by dissociation of GDP. Addition of RGS1 alone stimulated basal GTPase activity only modestly. This effect can best be explained by the influence of RGS1 on activated G-proteins present in unstimulated cell membranes, which accounts for the basal GTPase activity in this assay.

For the subsequent experiments a 10-min incubation period was chosen, which is well within the linear response of this assay. The data shown in Fig. 5A illustrate that stimulation of GTPase activity by fMLP in membranes from Bt₂cAMP-treated U937 cells is dose-dependent as is the influence of RGS1 on the basal GTPase activity. Experiments using a fixed concentration of RGS1 and varying concentrations of fMLP or fixed concentrations of fMLP and varying concentrations of RGS1 demonstrated a dose-dependent increase of RGS1 mediated GAP activity.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   fMLP induced GTPase activity and RGS1 GAP activity in U937 cells is receptor-dependent. Increasing amounts of fMLP (0, 10 and 100 nM, and 1 µM); GST-RGS1 (0, 10 and 100 nM, and 1 µM); fMLP (0, 10 and 100 nM, and 1 µM) in the presence of 1 µM GST-RGS1; GST-RGS1 (0, 10 and 100 nM, and 1 µM) in the presence of 1 µM fMLP were used in the GTPase assay. Basal and receptor-induced GTPase activity was determined after 10 min. Basal activity (no addition) was subtracted from stimulated GTPase activity. A, membranes from Bt2cAMP-treated U937 cells; fMLP-induced GTPase activity and RGS1-mediated GAP activity is concentration-dependent. Treatment with 100 ng/ml pertussis toxin (PTX) for 8 h before membrane preparation eliminates fMLP-stimulated, RGS1-stimulated, and fMLP/RGS1-stimulated GTPase activity. Addition of RGS1 antiserum inhibits RGS1-mediated GAP activity (RGS1 AS); heat treatment abolishes GAP activity of RGS1 (h.i.RGS1). B, membranes from untreated U937 cells.

Addition of RGS1 antibodies or prior heat treatment of RGS1 abolished GAP activity. Stimulation of GTPase activity by fMLP as well as ligand-specific GAP activity of RGS1 was sensitive to pertussis toxin (see Fig. 5A) suggesting that the GTPase activity measured is G_i- and/or G_o-dependent (38). No significant effect of fMLP or RGS1 on GTP hydrolysis was observed when using cell membranes from native U937 cells, indicating that Bt₂cAMP-induced expression of fMLP receptor is a strict requirement (Fig. 5B).

To exclude a possible influence of the large GST mass (27.5 kDa) on the functional activity of the RGS1 fusion protein used in our investigations, a fusion protein with a smaller tag (6xHis, 0.84 kDa) was generated. The GAP activity of the His-tagged fusion protein was found to be identical to that of the GST-RGS1 fusion protein. As shown in Fig. 6, both proteins showed a saturation of their GAP activity at 300 nM for fMLP-induced G phosphorylation. Also shown is a comparison with His-tagged RGS4. Compared with RGS1, RGS4 is less effective in GTP hydrolysis in quantitative terms with GAP activity saturating at 100 nM. The observed saturation of RGS4 GAP activity at 100 nM in our membrane-based signaling assay is well within the range of K_m values determined previously, i.e. for RGS4-mediated GAP activity in the M1 receptor-G_q system (29) and for RGS4-mediated inhibition of phospholipase C1 activity (28, 29).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Dose-response relationship of RGS1 and RGS4 GAP activity. GTPase activity was determined in plasma membranes (3 µg) from U937 cells treated with Bt2cAMP. Increasing amounts of His-RGS1 (0, 10, 100, 300, and 1000 nM); GST-RGS1 (0, 10, 100, 300, and 1000 nM); His-RGS4 (0, 10, 100, 300, and 1000 nM) in the presence of 1 µM fMLP were used in the GTPase assay. Basal activity (no addition) was subtracted from stimulated GTPase activity. The data shown are means ± S.D. of three individual experiments.

Given the effect of RGS1 on fMLP receptor-induced GTP hydrolysis, it was important to rule out that RGS1 affects guanine nucleotide exchange by causing the G subunit to release GDP and to bind GTP. RGS1 did not influence basal nor fMLP receptor-stimulated binding of the poorly hydrolyzable GTP analogue GTPS to cell membranes (Fig. 7, only results for the GST-RGS1 fusion protein are shown, identical results were obtained with His-RGS1 fusion protein). As the rate of pseudoirreversible binding of GTPS to G subunits is limited by dissociation of GDP (39, 40), these results indicate that RGS1 does not act as stimulator of guanine nucleotide dissociation.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   RGS1 does not affect ligand-dependent [35S]GTPS binding. [35S]GTPS binding was determined at various time points (0, 2.5, 5, and 10 min) after addition of 1 µM fMLP; 1 µM GST-RGS1; and 1 µM fMLP/1 µM GST-RGS1. Nonspecific binding (in the presence of 100 µM GTPS) was less than 1% of the total binding. The data shown are means ± S.D. of three individual experiments, each consisting of duplicate determinations.

RGS1 Is a General GAP Protein for Chemotactic Receptors-- The data shown above represent the first demonstration of alteration of ligand-induced GTPase activity by addition of an RGS protein to an endogenous, membrane-bound signaling system. We have tested the generality of this observation by examining the effects of exogenously added RGS1 on fMLP-induced GTPase activity in membranes from Bt₂cAMP-treated THP1 cells and by investigating other chemoattractants.

fMLP and RGS1 alone modestly affected GTPase activity in membranes from Bt₂cAMP-treated THP1 cells, e.g. addition of 1 µM fMLP resulted in hydrolysis of 32 pmol of GTP/min/mg of membrane protein and addition of 1 µM RGS1 led to hydrolysis of 38 pmol of GTP/min/mg of membrane protein. In contrast, RGS1 (1 µM) drastically accelerated fMLP (1 µM)-stimulated GTP hydrolysis resulting in hydrolysis of 339 pmol of GTP/min/mg of membrane protein (Fig. 8A).

View larger version (31K): [in this window] [in a new window]   Fig. 8.   RGS1 is a general GAP protein for chemotactic receptors. A, RGS1 GAP activity on fMLP-stimulated GTP hydrolysis in plasma membranes from Bt2cAMP-treated THP1 cells. Increasing amounts of fMLP (0, 10 and 100 nM, and 1 µM), GST-RGS1 (0, 10 and 100 nM, and 1 µM), fMLP (0, 10 and 100 nM, 1 µM) in the presence of 1 µM GST-RGS1, and GST-RGS1 (0, 10 and 100 nM, 1 µM) in the presence of 1 µM fMLP were used. GTPase activity was determined after 10 min; basal GTPase activity (no addition) was subtracted from GTPase activity after stimulation. B, RGS1 acts as GTPase-activating protein for fMLP, C5a, and LTB4 chemoattractant receptors in membranes of Bt2cAMP-treated U937 cells. The basal and receptor-stimulated GTPase activity was determined after 10 min. The basal GTPase activity (no addition) was subtracted from the stimulated GTPase activity. GST-RGS1 and the ligands indicated (fMLP, C5a, and LTB4) were each used in a concentration of 1 µM. The means ± S.D. of six independent experiments are shown.

Bt₂cAMP-treated U937 cells are known to express a variety of chemoattractant receptors (41, 42). As shown in Fig. 8B, stimulation with C5a (1 µM) and LTB4 (1 µM) resulted in a significant GTPase activity (100-150 pmol of GTP/min/mg of membrane protein). Addition of RGS1 markedly stimulated chemoattractant-induced GTPase activity resulting in hydrolysis of 782 pmol of GTP/min/mg of membrane protein for C5a/RGS1 and 336 pmol of GTP/min/mg of membrane protein for LTB4/RGS1 (Fig. 8B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In our search for genes that are expressed during maturation of monocytes, we isolated RGS1. RGS proteins were initially described as negative regulators of signal transduction in yeast and Caenorhabditis elegans, with actions likely to be exerted at the level of the receptor or the G-protein  subunit (18, 21, 22). To date, biochemical studies on the action of RGS proteins have been mostly performed using purified recombinant G subunits. These in vitro experiments have demonstrated that several RGS proteins are effective GAPs for G subunits of the G_i and the G_q family (19, 23-26).

RGS function was previously investigated by using purified, GTP-loaded G subunits in single turnover assays (19, 24); transfected mammalian cells stably expressing an RGS protein and an upstream (receptor) or downstream (kinase) signaling component (17, 43); phospholipid vesicles reconstituted with heterotrimeric G-proteins and an appropriate guanine nucleotide exchanger (receptor) to regenerate substrate for steady-state GTPase assays (by catalyzing dissociation of the product GDP) (29).

Our experiments demonstrate the stimulation of chemoattractant-induced GTPase activity by addition of an RGS protein to an endogenous, membrane-bound signaling system. To our knowledge our results provide the first direct evidence for a catalytic activation of the endogenous GTPase activity of G subunits by an RGS protein using ligand-stimulated receptors in an endogenous membrane-bound signaling system. Our experiments indicate that purified RGS1 can reassociate with cytoplasmic membranes and regulate G subunits appropriately. As an increase in GTP hydrolysis, as measured in our assay, can be due to stimulation of GTPase activity or to stimulation of guanine nucleotide dissociation or both, it was important to determine whether RGS1 affects GDP-GTP exchange. RGS1 did not influence the basal or fMLP-stimulated binding of the poorly hydrolyzable GTP analogue GTPS excluding the possibility that RGS1 acts as a guanine nucleotide dissociation stimulator. The effect of RGS1 on the steady-state GTPase rate is consistent with its observed activity in previous single turnover assays. This effect of RGS1 is exerted only at the hydrolytic step, as RGS1 did not affect binding of GTPS. In the absence of agonist, RGS1 had little effect on the GTPase activity of isolated cell membranes.

It has been proposed that RGS proteins, e.g. RGS8, function not only as GAP but also as an on-rate accelerator, for example in promoting GDP-GTP exchange (25). However, as the estimation of GDP-GTP exchange rate was done in the absence of stimulation by an activated receptor, it was not possible to address this question experimentally. In the case of RGS1, the GTPS binding results presented herein demonstrate that RGS1 does not act as on-rate accelerator.

We found that RGS1 is membrane-associated. Both endogenous RGS1 (TPA-treated THP1 cells) and transfected RGS1 (expressed in CHO cells) were found mainly in the membrane fraction of crude cellular extracts. By immunofluorescence studies using confocal laser scan microscopy most of the RGS1 protein localized to the cell membrane. The identical subcellular distribution observed for the tagged and for endogenous RGS1 demonstrates that the tag does not affect the cellular localization of the fusion protein. Resistance to stripping with 0.1 M Na₂CO₃ (35) and susceptibility of RGS1 in the membrane fraction to digestion with proteinase K indicates that RGS1 is a membrane-associated protein that is faced to the cytoplasm.

The subcellular localization of RGS proteins is likely to vary. Some RGS proteins are predicted to be extremely hydrophilic proteins that are likely to be found in the cytoplasm. Previously, two other RGS proteins, GAIP and RGS3, were found to pellet in the membrane fraction of crude cellular extracts, but no localization to specific membranes was provided (20, 44). GAIP, expressed in COS cells by transient transfection, was found to be membrane-anchored by palmitoylation (20). Plasma membrane localization was also found to be required for RGS4 function in Saccharomyces cerevisiae (45). RGS1 lacks a signal peptide and an evident transmembrane domain but contains three potential N-myristoylation sites (46, 47) at amino acid positions 3, 15, and 51.

The assignment of specific receptors to specific G-proteins and defined RGS proteins is one of the problems encountered in the study of G-protein signal transduction. Although rapid progress has been made in dissecting the biochemical mechanisms by which RGS proteins stimulate GTPase activity of G-protein  subunits, less is understood about RGS-mediated regulation of G-protein  phosphorylation in response to ligands. In single turnover assays several RGS proteins were able to stimulate GTP hydrolysis by several members of the G_i subfamily of G-protein  subunits. Under the conditions assayed, RGS proteins have shown little ability to distinguish among different members of the G_i subfamily. RGS proteins may thus have little specificity in their interaction with G-proteins, and specificity of function among RGS proteins would have to rely on their different expression pattern. As the activity and specificity of RGS proteins has been suggested to possibly include ancillary molecules (48, 49), it is possible that the specificity of RGS proteins may be different in the presence of an activated receptor. The herein described assay that allows determination of RGS-dependent GAP activity in ligand-stimulated plasma membranes might be a useful tool to address these questions in more detail.

Chemotaxis is crucial for leukocytes to migrate to sites of inflammation and infection, and a large number of chemotactic peptides and lipids have been demonstrated to be chemoattractants (50). The receptors for all known chemoattractants, including the chemokines, are members of the seven-transmembrane domain superfamily and couple to a variety of G-protein  subunits (3).

The finding that RGS1 is expressed in cells of the monocyte lineage prompted us to investigate a possible involvement of RGS1 in the chemoattractant signaling pathway. Immature histiocytic lymphoma U937 cells can be differentiated by a variety of stimuli to a more macrophage-like phenotype, e.g. receptors for the chemoattractants C5a, fMLP, and LTB4 are induced by treatment with the membrane-permeable cAMP analogue Bt₂cAMP. Bt₂cAMP treatment increases the number of chemoattractant receptors in U937 cells from an undetectable level in unstimulated cells to numbers of 30,000 receptor molecules (fMLP) and 100,000 receptor molecules (C5a) per cell, respectively (41, 42). Investigation of ligand-induced GTP hydrolysis using membrane preparations from Bt₂cAMP-treated U937 cells demonstrated that GAP activity of RGS1 is not limited to fMLP but is observed with a variety of other chemoattractants. Significant stimulation of GTP hydrolysis by RGS1 was observed with C5a and LTB4. Receptors for C5a, LTB4, and fMLP all interact with G_i3 and G_i2 (3). Investigation of Bt₂cAMP-treated THP1 cells demonstrated that our observations are not limited to U937 cells but a more general characteristic of chemoattractant signaling in monocytic cells. Given the function of RGS proteins as GAP proteins, these experiments suggest that RGS1 deactivates the chemotactic response. More recent data confirm a function for RGS1 in deactivation of chemotaxis in a system using transiently transfected lymphoid cells (51).

Mammalian RGS proteins are presumed to play an important role in the down-regulation or desensitization of G-protein-mediated signaling pathways. Technical complexities have delayed examination of RGS proteins with receptor-directed activation of G subunits. The availability of individual ligands in pure form, together with the sensitive methodology to assay RGS-mediated GTPase activity in ligand-stimulated cell membranes as outlined in this paper, should allow these questions to be addressed in more detail.

	ACKNOWLEDGEMENTS

We thank Manfred Rohde (GBF, Braunschweig, Germany) for performing laser scan microscopy, Ken Blumer and Ned Watson for generously providing the His-RGS4 vector (r(His)₆RGS4 in pET15b).

	FOOTNOTES

^* This study was supported by the Sonderforschungsbereich 244 "Chronische Entzündung" of the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Hermann and Lilly Schilling professor. To whom correspondence should be addressed. Tel.: 49-511-532 4348; Fax: 49-511-532 4366; E-mail: boettger.erik@mh-hannover.de.

	ABBREVIATIONS

The abbreviations used are: fMLP, N-formyl-methionyl-leucyl-phenylalanine; LTB4, leukotriene B4; GAP, GTPase-activating protein; Bt₂cAMP, dibutyryl cyclic AMP; GTPS, guanosine 5'-3-O-(thio)triphosphate; TPA, 12-tetradecanoyl-phorbol-13-acetate; IFN, interferon-; GST, glutathione S-transferase; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; mAb, monoclonal antibody; PBS, phosphate-buffered saline; RT, room temperature; App(NH)P, adenyl-5'-yl ,-imidodiphosphate; aa, amino acid. GAIP, G-interacting protein; MBP, maltese-binding protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES