Why affinity, functional potency and biased signalling require different measurements.
Binding and function
A binding assay examines interaction with a receptor under specified conditions. A functional assay examines a downstream cellular response. Because they measure different events, they should not be treated as interchangeable evidence.
The system shapes the result
Receptor density, coupling efficiency and readout dynamic range can shift the observed curve. Selectivity should therefore be reported with the expression system, incubation time, reference agonist and normalisation method.
Comparing with fitness for purpose
To compare three receptors, the procedure needs adequate sensitivity and repeatability in each system. Controls and system-suitability criteria should be set before analysis. Validation is always linked to the intended purpose.