Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta2 adrenergic receptor (CAM) is characterized by structural instability and an exaggerated conformational response to ligand binding. The structural instability of CAM could be demonstrated by a 4-fold increase in the rate of denaturation of purified receptor at 37 degrees C as compared with the wild type receptor. Spectroscopic analysis of purified CAM labeled with the conformationally sensitive and cysteine-reactive fluorophore, N,N'dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We propose that the mutation that confers constitutive activity to the beta2 adrenergic receptor removes some stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and making the receptor more susceptible to denaturation.