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. 2008 Sep;378(3):261-74.
doi: 10.1007/s00210-008-0313-8. Epub 2008 Jun 4.

Functional reconstitution of the human chemokine receptor CXCR4 with G(i)/G (o)-proteins in Sf9 insect cells

Patrick Kleemann  1 , Dan Papa, Sandy Vigil-Cruz, Roland Seifert
Affiliations

Functional reconstitution of the human chemokine receptor CXCR4 with G(i)/G (o)-proteins in Sf9 insect cells

Patrick Kleemann et al. Naunyn Schmiedebergs Arch Pharmacol. 2008 Sep.

Abstract

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) binds to the chemokine receptor CXCR4 that couples to pertussis toxin-sensitive G-proteins of the G(i)/G(o)-family. CXCR4 plays a role in the pathogenesis of autoimmune diseases, human immunodeficiency virus infection and various tumors, fetal development as well as endothelial progenitor and T-cell recruitment. To this end, most CXCR4 studies have focused on the cellular level. The aim of this study was to establish a reconstitution system for the human CXCR4 that allows for the analysis of receptor/G-protein coupling at the membrane level. We wished to study specifically constitutive CXCR4 activity and the G-protein-specificity of CXCR4. We co-expressed N- and C-terminally epitope-tagged human CXCR4 with various G(i)/G(o)-proteins and regulator of G-protein signaling (RGS)-proteins in Sf9 insect cells. Expression of CXCR4, G-proteins, and RGS-proteins was verified by immunoblotting. CXCR4 coupled more effectively to Galpha(i1) and Galpha(i2) than to Galpha(i3) and Galpha(o) and insect cell G-proteins as assessed by SDF-1alpha-stimulated high-affinity steady-state GTP hydrolysis. The RGS-proteins RGS4 and GAIP enhanced SDF-1alpha-stimulated GTP hydrolysis. SDF-1alpha stimulated [(35)S]guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) binding to Galpha(i2). RGS4 did not enhance GTPgammaS binding. Na(+) salts of halides did not reduce basal GTPase activity. The bicyclam, 1-[[1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100), acted as CXCR4 antagonist but was devoid of inverse agonistic activity. Halides reduced the maximum SDF-1alpha-stimulated GTP hydrolysis in the order of efficacy I(-) > Br(-) > Cl(-). In addition, salts reduced the potency of SDF-1alpha at activating GTP hydrolysis. From our data, we conclude the following: (1) Sf9 cells are a suitable system for expression of functionally intact human CXCR4; (2) Human CXCR4 couples effectively to Galpha(i1) and Galpha(i2); (3) There is no evidence for constitutive activity of CXCR4; (4) RGS-proteins enhance agonist-stimulated GTP hydrolysis, showing that GTP hydrolysis becomes rate-limiting in the presence of SDF-1alpha; (5) By analogy to previous observations made for the beta(2)-adrenoceptor coupled to G(s), the inhibitory effects of halides on agonist-stimulated GTP hydrolysis may be due to increased GDP-affinity of G(i)-proteins, reducing the efficacy of CXCR4 at stimulating nucleotide exchange.

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Figures

Fig. 1
Fig. 1. Analysis of the expression of CXCR4 and Gα subunits in Sf9 membranes
SDS-PAGE and immunoblots were performed as described in Materials and Methods. Unless stated otherwise in panel B, 10 μg of membrane protein were loaded onto each lane. Numbers on the left indicate molecular masses (kDa) of marker proteins. Numbers below immunoblots designate the specific membrane studied (see Table 1 for specific protein composition). Shown are the immunoblots of gels containing 12% (m/v) acrylamide. A, anti-FLAG Ig with CXCR4-expressing membranes; B, anti-FLAG Ig with β2AR standard membrane (7.5 pmol/mg as assessed by [3H]dihydroalprenolol saturation binding; 2–15 μg of protein/lane) and one representative CXCR4 membrane; C, anti-Gγicommon Ig with CXCR4 membranes; D, anti-Gαi1,2 Ig with CXCR4 membranes; E, anti-Gαo Ig with CXCR4 membranes.
Fig. 2
Fig. 2. Analysis of the expression of CXCR4, Gα subunits, Gβ subunits and RGS-proteins in Sf9 membranes
SDS-PAGE and immunoblots were performed as described in Materials and Methods. Unless stated otherwise in panel A, 10 μg of membrane protein were loaded onto each lane. Numbers on the left indicate molecular masses (kDa) of marker proteins. Numbers below immunoblots designate the specific membrane studied (see Table 1 for specific protein composition). Shown are the immunoblots of gels containing 12% (m/v) acrylamide. A, anti-Gαi1,2 Ig with β2AR-Gαi2 standard membranes (3.0 pmol/mg as assessed by [3H]dihydroalprenolol saturation binding; 20–250 μg of protein/lane) and representative CXCR4-expressing membranes; B, anti-Gβcommon Ig with CXCR4 membranes; C, anti-hexahistidine Ig with CXCR4 membranes; D, anti-RGS4 Ig with CXCR4 membranes; E, anti-RGS4 plus anti-GAIP Ig with CXCR4 membranes.
Fig. 3
Fig. 3. Comparison of basal and SDF-1α-stimulated GTPase activity in various CXCR4-expressing Sf9 membranes
High-affinity GTPase activity was determined as described in Materials and Methods. Numbers below columns designate the specific membrane studied (see Table 1 for specific protein composition). A, absolute GTPase activities under basal conditions (0.2% (m/v) BSA) or in the presence of a maximally stimulatory concentration of SDF-1α (50 nM). B, relative stimulatory effect of SDF-1α. Data shown are the means ± SD of three independent experiments performed in duplicates. The statistical significance of the effects of RGS-proteins RGS4 and GAIP versus control on SDF-1α-stimulated GTP hydrolysis was assessed using the t-test. *, p < 0.05.
Fig. 4
Fig. 4. Concentration/response curves for the effects of SDF-1α and AMD3100 on GTPase activity in Sf9 membranes expressing CXCR4
High-affinity GTPase activity was determined as described in Materials and Methods in Sf9 membrane 1182 (CXCR4 + Gαi2 + Gβ1γ2). A, Effects of SDF-1αand AMD3100 on basal GTPase activity. B, Inhibitory effect of AMD3100 on GTPase activity stimulated by SDF-1α (10 nM). Data shown are the means ± SD of a representative experiment performed in triplicates. Similar results were obtained in two independent experiments with different membrane preparations.
Fig. 5
Fig. 5. Concentration/response curves for the effects of various monovalent salts on basal and SDF-1-α-stimulated GTPase activity in Sf9 membranes expressing CXCR4
High-affinity GTPase activity was determined as described in Materials and Methods in Sf9 membrane 1182 (CXCR4 + Gαi2 + Gβ1γ2). Reaction mixtures contained either 0.2% (m/v) BSA (basal) or 50 nM SDF-1α. Additionally, reaction mixtures contained monovalent salts at the concentrations indicated on the abscissa. A, LiCl; B, NaCl; C, KCl; D, LiBr; E, NaBr; F, KBr; G, LiI; H, NaI; I, KI. Data shown are the means ± SD of a representative experiment performed in triplicates. Similar results were obtained in two independent experiments with different membrane preparations.
Fig. 6
Fig. 6. Concentration/response curves for the effects of SDF-1α on GTPase activity in Sf9 membranes expressing CXCR4 in the presence of various monovalent salts
High-affinity GTPase activity was determined as described in Materials and Methods in Sf9 membrane 1182 (CXCR4 + Gαi2 + Gβ1γ2). Reaction mixtures contained SDF-1α at various concentrations in the absence and presence of monovalent salts at various fixed concentrations (50 mM, 100 mM or 150 mM). A, effect of LiCl; B, effect of NaCl; C, effect of KCl. Data shown are the means ± SD of a representative experiment performed in triplicates. Similar results were obtained in two independent experiments with different membrane preparations.
Fig. 7
Fig. 7. Regulation of GTPγS binding by SDF-1α in Sf9 membranes expressing CXCR4
GTPγS binding was determined as described in Materials and Methods in Sf9 membrane 1182 (CXCR4 + Gαi2 + Gβ1γ2) and 1183 1182 (CXCR4 + Gαi2 + Gβ1γ2 + RGS4). A and B, time course of GTPγS binding. Reaction mixtures contained 1 μM GDP plus 0.4 nM [35S]GTPγS and 15 μg of membrane 1182 (A) or 1183 (B). Reactions were conducted for the periods of time indicated on the abscissa. Basal GTPγS binding was subtracted from GTPγS binding in the presence of SDF-1α. Thus, SDF-1α-stimulated GTPγS binding is shown. C and D, GTPγS saturation binding. Reaction mixtures contained 1 μM GDP plus 0.2–20 nM [35S]GTPγS as indicated on the abscissa and 15 μg of membrane 1182 (C) or 1183 (D). Reactions were conducted for 90 min. Basal GTPγS binding was subtracted from SDF-1α-stimulated GTPγS binding. Thus, SDF-1α-stimulated GTPγS binding is shown. Data shown are the means ± SD of three independent experiments performed in duplicates. Similar results were obtained in two independent experiments with different membrane preparations.
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