Detection of g protein coupled receptor mediated adenylyl cyclase activity by capillary electrophoresis using fluorescently labeled ATP.
A capillary electrophoresis (CE) laser-induced fluorescence (LIF) assay was developed for the detection of G protein coupled receptor mediated adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, cell membranes coexpressing the stimulatory G protein fused to the beta2 adrenergic receptor (beta2AR) and AC were incubated with BATP, the resultant mixture injected, and BATP separated from product BODIPY FL cAMP (BcAMP) by CE. AC activity was quantified by measuring the rate of BcAMP formation. beta2AR agonists isoproterenol and terbutaline increased basal AC activity with EC50s of 2.4 +/- 0.2 and 60 +/- 9 nM, respectively. The antagonist propranolol competed with terbutaline for beta2AR binding sites and expectedly right-shifted the terbutaline dose-response curve to 8 +/- 3 microM. The high sensitivity of the assay was demonstrated by detection of small changes in AC activity, with the partial agonist alprenolol increasing (22 +/- 1%) and the inverse agonist ICI 118,551 decreasing (19 +/- 2%) basal activity. The simplicity and automation of the CE-LIF assay offers advantages over the more traditional assay using radiochemical ATP and column chromatography.
Cunliffe
Jennifer M
JM
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Sunahara
Roger K
RK
Kennedy
Robert T
RT
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D019075:Electrophoresis, Capillary / Q000379:methods*; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018411:Spodoptera; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D043562:Receptors, G-Protein-Coupled; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases