Pathway experiments
Compare untreated cells with controls that establish the normal signal response. Inhibitors, loss-of-function mutations, and permanently active proteins can reveal a pathway order. A downstream activation that restores the response supports an upstream problem, but does not prove the exact defective molecule without additional evidence. Distinguish ligand binding from transduction and from the measured response. Use the simplest model consistent with all treatments, including negative controls.
A mutant fails to respond to hormone but responds to a membrane-permeable second-messenger analog. Its machinery downstream of the second messenger can still function.
A rescued response narrows possible defects; it does not magically identify a specific mutated gene.