Membrane protein microarrays

This paper describes the fabrication of microarrays consisting of G protein-coupled receptors (GPCRs) on surfaces coated with γ-aminopropylsilane (GAPS). Microspots of model membranes on GAPS-coated surfaces were observed to have several desired propertieshigh 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 (β1), 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: β1, β2, and α2A. When treated with fluorescently labeled CGP 12177, a cognate antagonist analogue specific to β-adrenergic receptors, binding was only observed to microspots of the β1 and β2 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 β2 receptor relative to that for the β1 receptor. The ability to estimate binding affinities of compounds using GPCR arrays was examined using a competitive binding assay with BT-NT and unlabeled nuerotensin on NTR1 arrays. The estimated IC50 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 technologythe lack of practical microarray-based methods for membrane proteins. Copyright © 2002 American Chemical Society.