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Functional reconstitution of the human chemokine receptor CXCR4 with Gi/Go-proteins in Sf9 insect cells

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Abstract

The chemokine stromal cell-derived factor-1α (SDF-1α) binds to the chemokine receptor CXCR4 that couples to pertussis toxin-sensitive G-proteins of the Gi/Go-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 Gi/Go-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 Gαi1 and Gαi2 than to Gαi3 and Gαo and insect cell G-proteins as assessed by SDF-1α-stimulated high-affinity steady-state GTP hydrolysis. The RGS-proteins RGS4 and GAIP enhanced SDF-1α-stimulated GTP hydrolysis. SDF-1α stimulated [35S]guanosine 5′-[γ-thio]triphosphate (GTPγS) binding to Gαi2. RGS4 did not enhance GTPγS 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-1α-stimulated GTP hydrolysis in the order of efficacy I− > Br− > Cl−. In addition, salts reduced the potency of SDF-1α 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 Gαi1 and Gαi2; (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-1α; (5) By analogy to previous observations made for the β2-adrenoceptor coupled to Gs, the inhibitory effects of halides on agonist-stimulated GTP hydrolysis may be due to increased GDP-affinity of Gi-proteins, reducing the efficacy of CXCR4 at stimulating nucleotide exchange.

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Abbreviations

AMD3100:

1-[[1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane

BSA:

bovine serum albumin

β2AR:

β2-adrenoceptor

GPCR:

G-protein-coupled receptor

GTPγS:

guanosine 5′-[γ-thio]triphosphate

GAIP:

Gα-interacting protein

RGS protein:

Regulator of G-protein Signalling protein

SDF-1α:

stromal cell-derived factor-1α

Sf9 cells:

Spodoptera frugiperda pupal ovary cells

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Acknowledgments

We thank Dr. Erich Schneider for stimulating discussions as well as Mrs. Astrid Seefeld and Gertraud Wilberg for expert technical assistance. Thanks are also due to the reviewers of the paper for their constructive critique and suggestions.

This work was supported by the Research Training Program (Graduiertenkolleg) GRK 760 “Medicinal Chemistry: Molecular Recognition—Ligand–Receptor Interactions” of the Deutsche Forschungsgemeinschaft, the “International Study and Training Partnerships (ISAP) Program” of the Deutscher Akademischer Austauschdienst and by the National Institutes of Health COBRE Award 1 P20 RR15563 and matching support from the State of Kansas.

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Author notes

  1. Patrick Kleemann

    Present address: 3. Medizinische Klinik und Poliklinik, Hämatologie und Onkologie, Johannes-Gutenberg-Universität Mainz, D-55131, Mainz, Germany

  2. Dan Papa

    Present address: Biopharmaceutical Analysis, Aptuit, Kansas City, MO, 64137, USA

Authors and Affiliations

  1. Lehrstuhl für Pharmakologie und Toxikologie, Institut für Pharmazie, Universität Regensburg, Universitätsstraße 31, D-93053, Regensburg, Germany

    Patrick Kleemann & Roland Seifert

  2. Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS, 66045, USA

    Dan Papa

  3. Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, 66045, USA

    Sandy Vigil-Cruz

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  1. Patrick Kleemann
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Correspondence to Roland Seifert.

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Patrick Kleemann and Dan Papa contributed equally to this work.

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Kleemann, P., Papa, D., Vigil-Cruz, S. et al. Functional reconstitution of the human chemokine receptor CXCR4 with Gi/Go-proteins in Sf9 insect cells. Naunyn-Schmied Arch Pharmacol 378, 261–274 (2008). https://doi.org/10.1007/s00210-008-0313-8

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