Kleemann, Patrick and Papa, Dan and Vigil-Cruz, Sandy and Seifert, Roland (2008) Functional reconstitution of the human chemokine receptor CXCR4 with G(i)/G(o)-proteins in Sf9 insect cells. NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 378 (3). pp. 261-274. ISSN 0028-1298, 1432-1912
Full text not available from this repository. (Request a copy)Abstract
The chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha) 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 G alpha(i1) and G alpha(i2) than to G alpha(i3) and G alpha(o) and insect cell G-proteins as assessed by SDF-1 alpha-stimulated high-affinity steady-state GTP hydrolysis. The RGS-proteins RGS4 and GAIP enhanced SDF-1 alpha-stimulated GTP hydrolysis. SDF-1 alpha stimulated [S-35]guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) binding to G alpha(i2). RGS4 did not enhance GTP gamma S 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-1 alpha-stimulated GTP hydrolysis in the order of efficacy I- > Br- > Cl-. In addition, salts reduced the potency of SDF-1 alpha 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 G alpha(i1) and G alpha(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-1 alpha; (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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | FORMYL PEPTIDE RECEPTOR; BINDING REGULATORY PROTEINS; CONSTITUTIVE ACTIVITY; SIGNAL-TRANSDUCTION; LIGAND-BINDING; ANTAGONISTS; EXPRESSION; BETA(2)-ADRENOCEPTOR; FACTOR-1-ALPHA; G(Q)-PROTEINS; chemokine receptor; stroma cell-derived factor-1 alpha; G(i)/G(o)-proteins; RGS-proteins; GTPase activity; Sf9 insect cells |
| Subjects: | 600 Technology > 615 Pharmacy |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy > Pharmacology and Toxicology (Prof. Schlossmann, formerly Prof. Seifert) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 26 Oct 2020 09:53 |
| Last Modified: | 26 Oct 2020 09:53 |
| URI: | https://pred.uni-regensburg.de/id/eprint/30411 |
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