Development of surface-based assays for transmembrane proteins: Selective immobilization of functional CCR5, a G protein-coupled receptor

doi:10.1016/j.ab.2005.10.025

Analytical Biochemistry

Volume 349, Issue 2, 15 February 2006, Pages 247-253

https://doi.org/10.1016/j.ab.2005.10.025 Get rights and content

Abstract

A general method to develop surface-based assays for transmembrane (TM) receptor function(s) without the need to isolate, purify, and reconstitute the proteins is presented. Based on the formation of an active surface that selectively immobilizes membrane vesicles, the method is illustrated using the chemokine receptor CCR5, a member of the largest family of cell surface eukaryotic TM proteins, the G protein-coupled receptors (GPCRs). The method begins with a protein-resistant surface containing a low percentage (1–5%) of surface-bound biotin on gold as the initial template. Surface plasmon resonance (SPR) data show specific immobilization of functional CCR5 after the initial template is activated by immobilization of rho 1D4 antibody, an anti-rhodopsin monoclonal antibody specific for the carboxyl terminal nine amino acids on bovine rhodopsin that had been engineered into the carboxyl terminus of CCR5, and exposure to vesicles obtained from mammalian cells transfected with a synthetic human CCR5 gene. Activation of the initial template is effected by sequential immobilization of avidin, which binds to the biotin in the initial template, a biotinylated goat anti-mouse immunoglobulin G (Bt-IgG), which binds to the avidin binding sites distal to the surface and the F_c portion of the rho 1D4 antibody through its F_ab region(s) and finally rho 1D4. This approach establishes a broad outline for the development and application of various assays for CCR5 functions. SPR data also showed that vesicle immobilization could be achieved through an integrin–integrin antibody interaction after activation of the initial template with a goat anti-human integrin β1 antibody. These results suggest that the generic nature of the initial platform and flexibility of the subsequent surface activation for specific immobilization of membrane vesicles can be applied to the development of assays for other GPCRs or TM receptors for which antibodies are available or can be engineered to contain a particular antibody epitope.

Section snippets

Materials and methods

Avidin and steptavidin were purchased from Sigma–Aldrich Chemical (St. Louis, MO, USA), biotinylated IgG antibodies were obtained from Chemicon International (Temecula, CA, USA), and the anti-human integrin β1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). n-Undecylenic bromide was purchased from Pfaltz & Bauer (Waterbury, CT, USA). All other chemicals were purchased from Aldrich Chemical (Milwaukee, WI, USA). Tetrahydrofuran and hexamethylphosphoramide were dried

Results and discussion

Fig. 1A shows the SPR shifts obtained for the activation of the initial template by sequential immobilization of avidin, a biotin-conjugated goat anti-mouse IgG (Bt-IgG), and rho 1D4, an anti-rhodopsin antibody that recognizes the modified carboxyl-terminal region on CCR5. Immobilization and saturation are rapid for all steps, with essentially diffusion-limited kinetics for avidin. The kinetics of the Bt-IgG adsorption is slower than that of avidin, presumably due to its larger size (Bt-IgG ∼ 150

Conclusions

Highly reproducible selective attachment of membrane vesicles containing heterologously expressed functional CCR5 and endogenous integrin has been demonstrated using an initial template containing 1–5% biotin in a protein-resistant matrix. Selectivity of membrane vesicle immobilization is accomplished by sequential treatment with avidin or streptavidin, a biotinylated antibody, and a receptor (protein)-specific antibody treatment of the initial template. Considering the complexity of the

Acknowledgments

This work was supported in part by National Institutes of Health grant EY13286 to Kevin Ridge. Portions of this work were presented by Evan Karlik at the 2003 Montgomery County (MD) Area Science Fair (Life Sciences Grand Prize recipient) and at the 2003 International Science and Engineering Fair (Cleveland, OH). Karlik was also a semifinalist in the 2003 Intel Science Talent Search. Certain commercial equipment, instruments, and/or materials are identified in this article to specify the

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¹: Present address: University of Texas Health Science Center-Houston, Center for Membrane Biology, Department of Biochemistry and Molecular Biology, Houston, TX 77030, USA.

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Analytical Biochemistry

Abstract

Section snippets

Materials and methods

Results and discussion

Conclusions

Acknowledgments

References (10)

Characterization of biomimetic surfaces formed from cell membranes

Biophys. J.

Light stable rhodopsin: I. A rhodopsin analog reconstituted with a nonisomerizable 11-cis retinal derivative

J. Biol. Chem.

SPR studies of the non-specific adsorption kinetics of human IgG and BSA on gold surfaces modified by self-assembled monolayers (SAMs)

J. Colloid Interface Sci.

Cell membrane hybrid bilayers containing the G-protein-coupled receptor CCR5

Anal. Biochem.

Isostructural self-assembled monolayers: II. Methyl 1-(3-mercaptopropyl)oligo(ethylene oxide)s

Langmuir

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Surface plasmon resonance analysis of seven-transmembrane receptors

Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery

Fabrication of protein-anchoring surface by modification of Sio<inf>2</inf> with liposomal bilayer

Synthesis of Unsymmetrical Ketones Using Chelation-Controlled Sequential Substitution of N-Triazinylamide/Weinreb Amide by Organometallic Reagents