Multiplexing G protein-coupled receptors in microarrays: a radioligand-binding assay.
Multiplexing of G protein-coupled receptors (GPCRs) in microarrays promises to increase the efficiency, reduce the costs, and improve the quality of high-throughput assays. However, this technology is still nascent and has not yet achieved the status of "high throughput" or laid claim to handling a large set of receptors. In addition, the technology has been demonstrated only when using fluorescent ligands to detect binding, limiting its application to a subset of GPCRs. To expand the impact of multiplexing on this receptor class, we have developed a radiometric approach to the microarray assay. In these studies, we considered two receptors in the alpha-adrenergic receptor family, alpha2A and alpha2C, and the 125I-labeled agonist clonidine. We demonstrate that microarrays of these receptors can be readily detected (signal/noise ratio approximately 160) using a Typhoon 9210 PhosphorImager. In addition, biochemical characterization shows that ligand-binding profiles and selectivity are preserved with the selective antagonists BRL44408 and ARC239. Importantly, these microarrays use approximately 200- to 400-fold less membrane preparation required by conventional assay methods and allow two or more receptors to be assayed in an area equivalent to a standard well of a microtiter plate. The impact of this approach on screening in drug discovery is discussed.
Posner
Bruce
B
Pfizer, Groton, CT 06340, USA.
Hong
Yulong
Y
Benvenuti
Eric
E
Potchoiba
Michael
M
Nettleton
Dave
D
Lui
Li
L
Ferrie
Ann
A
Lai
Fang
F
Fang
Ye
Y
Miret
Juan
J
Wielis
Chris
C
Webb
Brian
B
D000317:Adrenergic alpha-Antagonists / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D003000:Clonidine / Q000032:analysis; D006224:Cricetinae; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D054833:Isoindoles; D007546:Isoquinolines / Q000737:chemistry; D010879:Piperazines / Q000737:chemistry; D040081:Protein Array Analysis / Q000191:economics / Q000379:methods*; D011485:Protein Binding; D011869:Radioligand Assay / Q000295:instrumentation* / Q000379:methods*; D011941:Receptors, Adrenergic / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*
D000317:Adrenergic alpha-Antagonists; D007093:Imidazoles; D007211:Indoles; D054833:Isoindoles; D007546:Isoquinolines; D010879:Piperazines; D011941:Receptors, Adrenergic; D043562:Receptors, G-Protein-Coupled; C032994:AR-C239; D003000:Clonidine; C072125:BRL 44408