PI hydrolysis assays:
	These assays will be performed as previously detailed by us . In brief, 2 x 106 cells/24 well plate expressing the receptor of interest are plated into 24-well plates and over-night in complete medium. The next day the medium is switched to serum-free, inositol-free Dulbecco’s Modified Eagle Medium containing 1 uCi/ml 3H-inositol. After an overnight incubation, the cells are gently rinsed with Kreb-Bicarbonate buffer containing 15 mM LiCl and incubated for 60 min in the presence and absence of various concentrations of test agents. The KRB is aspirated and the reaction terminated by the addition of 1 ml of stop solution (MeOH:H20:HCl at 100:100:1). 3H-inositol monophosphate is then extracted, purified and quantified as previously detailed. Representative PI hydrolysis data: Representative Kact and %Vmax data for PI hydrolysis protocol: Template for Kact and %Vmax determinations in 24-well plate format: BLANK BLANK BLANK 10 mM STD #1 10 mM STD #1 10 mM STD #1 10 mM STD #2 10 mM STD #2 10 mM STD #2 10 mM UNK #1 10 mM UNK #1 10 mM UNK #1 10 mM UNK #2 10 mM UNK #2 10 mM UNK #2 10 mM UNK #3 10 mM UNK #3 10 mM UNK #3 10 mM UNK #4 10 mM UNK #4 10 mM UNK #4 10 mM UNK #5 10 mM UNK #5 10 mM UNK #5 For calculation of Kact and Vmax values, SigmaPlot V4.0 is used to fit the raw data to the following equation: V= Vmax * X + NSA         Kact + X   Where V=Activity at each concentration of X (compound of interest); X=concentration of compound of interest; Vmax=parameter to be estimated; Kact=parameter to be estimated; NSA=non-specific activity at baseline. We have used the procedure extensively to calculate Vmax and Kact values.
	cAMP Stimulation Assays:
	These assays will be performed as previously detailed by us 2 x 106 cell/24 well plate are grown overnight and the next day switched to serum-free medium. After a 4 hr incubation, they are exposed to varying concentrations of test agent, with a parallel set of assays being performed with the standard agonist of interest. After a 20 min incubation at 37 C, the reaction is terminated and cAMP accumulation measured using a commercially available kit as previously detailed. Representative cAMP stimulation data with Kact values and Hill Coefficients calculated: Template for Kact and %Vmax determinations in 24-well plate format: BLANK BLANK BLANK 10 mM STD #1 10 mM STD #1 10 mM STD #1 10 mM STD #2 10 mM STD #2 10 mM STD #2 10 mM UNK #1 10 mM UNK #1 10 mM UNK #1 10 mM UNK #2 10 mM UNK #2 10 mM UNK #2 10 mM UNK #3 10 mM UNK #3 10 mM UNK #3 10 mM UNK #4 10 mM UNK #4 10 mM UNK #4 10 mM UNK #5 10 mM UNK #5 10 mM UNK #5 For calculation of Kact and Vmax values, SigmaPlot V4.0 is used to fit the raw data to the following equation: V= Vmax * X + NSA          Kact + X   Where V=Activity at each concentration of X (compound of interest); X=concentration of compound of interest; Vmax=parameter to be estimated; Kact=parameter to be estimated; NSA=non-specific activity at baseline. We have used the procedure extensively to calculate Vmax and Kact values.
	Inhibition of cAMP accumulation for Gi coupled receptors.
	Inhibition of forskolin-stimulated adenylate cyclase will be performed as previously detailed by us . In brief, cells will be grown in 24-well plates as described above, switched to serum-free medium for 4 hr and then stimulated with forskolin in the presence and absence of test agents. cAMP accumulation will be measured with a commercially available kit. The % inhibition of forskolin-stimulated adenylate cyclase activity will then be calculated and the data fit to the Kact and Vmax equation. Representative cAMP inhibition data: Template for Kact and %Vmax determinations in 24-well plate format: BLANK BLANK BLANK 10 mM STD #1 10 mM STD #1 10 mM STD #1 10 mM STD #2 10 mM STD #2 10 mM STD #2 10 mM UNK #1 10 mM UNK #1 10 mM UNK #1 10 mM UNK #2 10 mM UNK #2 10 mM UNK #2 10 mM UNK #3 10 mM UNK #3 10 mM UNK #3 10 mM UNK #4 10 mM UNK #4 10 mM UNK #4 Forskolin Forskolin Forskolin For calculation of Kact and Vmax values, SigmaPlot V4.0 is used to fit the raw data to the following equation: V= Vmax * X + NSA          Kact + X   Where V=Activity at each concentration of X (compound of interest); X=concentration of compound of interest; Vmax=parameter to be estimated; Kact=parameter to be estimated; NSA=non-specific activity at baseline. We have used the procedure extensively to calculate Vmax and Kact values.
	TRANSPORTER ASSAYS:
	Template for Kact and %Vmax determinations in 24-well plate format: BLANK BLANK BLANK 10 mM STD #1 10 mM STD #1 10 mM STD #1 10 mM STD #2 10 mM STD #2 10 mM STD #2 10 mM UNK #1 10 mM UNK #1 10 mM UNK #1 10 mM UNK #2 10 mM UNK #2 10 mM UNK #2 10 mM UNK #3 10 mM UNK #3 10 mM UNK #3 10 mM UNK #4 10 mM UNK #4 10 mM UNK #4 10 mM UNK #5 10 mM UNK #5 10 mM UNK #5
	--Cells are seeded at 1x105 cells/ml into 24-well plates and grown overnight in medium containing dialyzed serum. The next day, cells are switched to a modified KRB buffer (see above) and pre-incubated for 20 min at 37 C. Cells are then incubated with 1 uM (final concentration) 3H-5HT, DA or NE for 4 min. The reaction is then terminated by aspiration and the cells washed three times with ice-cold incubation buffer. Cells are then dissolved with 500 ul of 0.5 N NaOH with 350 ul added to AQUASURE and radioactivity measured after chemilumiscence has decayed (generally overnight). --Representative Ki values for inhibition of 5-HT uptake found in Nakaki et al, 1995 and Palvimakki et al, 1996.
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