Membrane Protein Microarrays

Abstract

This paper describes the fabrication of microarrays consisting of G protein-coupled receptors (GPCRs) on surfaces coated with gamma-aminopropylsilane (GAPS). Microspots of model membranes on GAPS-coated surfaces were observed to have several desired properties-high mechanical stability, long range lateral fluidity, and a thickness corresponding to a lipid bilayer in the bulk of the microspot. GPCR arrays were obtained by printing membrane preparations containing GPCRs using a quill-pin printer. To demonstrate specific binding of ligands, arrays presenting neurotensin (NTR1), adrenergic (beta1), and dopamine (D1) receptors were treated with fluorescently labeled neurotensin (BT-NT). Fluorescence images revealed binding only to microspots corresponding to the neurotensin receptor; this specificity was further demonstrated by the inhibition of binding in the presence of excess unlabeled neurotensin. The ability of GPCR arrays to enable selectivity studies between the different subtypes of a receptor was examined by printing arrays consisting of three subtypes of the adrenergic receptor: beta1, beta2, and alpha2A. When treated with fluorescently labeled CGP 12177, a cognate antagonist analogue specific to beta-adrenergic receptors, binding was only observed to microspots of the beta1 and beta2 receptors. Furthermore, binding of labeled CGP 12177 was inhibited when the arrays were incubated with solutions also containing ICI 118551, and in a manner consistent with the higher affinity of ICI 118551 for the beta2 receptor relative to that for the beta1 receptor. The ability to estimate binding affinities of compounds using GPCR arrays was examined using a competitive binding assay with BT-NT and unlabeled neurotensin on NTR1 arrays. The estimated IC(50) value (2 nM) for neurotensin is in agreement with the literature; this agreement suggests that the receptor -G protein complex is preserved in the microspot. This first ever demonstration of direct pin-printing of membrane proteins and ligand-binding assays thereof fills a significant void in protein microchip technology--the lack of practical microarray-based methods for membrane proteins.

Microarrays to characterize protein interactions on a whole‐proteome scale

“…In Fig. 5 nal G-protein coupled receptors in a lipid bilayer can be reconstituted on glass slides and shown to bind to specific ligands [56]. These results clearly show the potential of protein arrays for high-throughput drug screening for membrane-associated proteins, usually considered to be the most intractable for these types of analyses.…”

Characterization of radioligand binding to a transmembrane receptor reconstituted into Lipobeads

“…bead). We therefore expect that 7 mg of dry beads will yield 0.035 cm 3 (35 lL) of hydrated beads, given that Lipobeads have a density of 1 g/cm 3 . Since the volume of a single bead is 5.2 Â 10 À7 cm 3 , based on a radius of 0.005 cm, the mixture must contain about 6.7 Â 10 4 beads, or 6-fold fewer than required to accommodate all available liposomes.…”

“…Transmembrane proteins therefore are often studied in preparations of protein-enriched membrane fragments and, upon solubilization, after subsequent reconstitution in liposomes [1,2]. To overcome the mechanical instability associated with these classical methods, and to facilitate ease of handling, supported phospholipid membranes on modified solid surfaces have been developed [3][4][5][6]. The solid surfaces used to support phospholipid membranes typically are made up of a flat sheet of glass, silica, or a surface coated with gold.…”

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

“…The authors were able to follow ligand binding, G protein activation, and receptor deactivation by surface plasmon resonance (SPR). Fang et al investigated the structure and properties of a G protein-coupled receptor embedded in a supported lipid bilayer on a microarray slide surface [81,82]. Lipid microspots of a mixture of vesicular solutions of lecitins and the membrane protein are stable on an aminopropyl silane coated surface.…”