Question

The type 1 angiotensin receptor (AT1R) mediates the important physiological actions of the peptide hormone angiotensin II to regulate blood pressure and water and salt balance. Research using mutated AT1Rs and substituted versions of angiotensin II, modified at key positions involved in receptor activation, have provided evidence for multiple functional receptor states for the AT1R. Outline the nature of this evidence, the experimental approaches, outcomes and interpretations, and rationalise the potential number and type of receptor states predicted from this data. Finally, speculate on how this information might be used to develop new drugs that selectively modulate the AT1R in disease.

Answer 1

The AT1R and angiotensin are the two main components of the Renin-Angiotensin system, which regulates blood pressure and water balance in the human body. The biochemistry involves a ligand-receptor interaction which triggers a series of intracellular reactions that ultimately cause the physiological changes as described.

Receptor - AT1R (angiotensin type I), ligand - angiotensin II.

The AT1R-angiotensin II binding can be quantified by using a radiolabeled angiotensin II molecule, which can be used to determine the IC₅₀ value (a measure of affinity) of the complex, through a saturation binding assay. This value will increase in the presence of various antagonists or blockers, which inhibit angiotensin II binding to the AT1R receptor. It can also change if the AT1R is mutated.

To obtain various conformational states of the free AT1R or AT1R-angiotensin II complex, their crystal must be obtained. The exact binding sites can be mapped from these crystal structures. Key residues involved in binding can be modified by site-directed mutagenesis to obtain mutants whose binding affinity can be determined by the saturation binding assay as described above. Moreover, a comparison of the free and ligand-bound AT1R structures will help to elucidate mechanisms of ligand-induced conformation change in the receptor, as well as the mechanism of 'activation' by the ligand.

If the receptor has a single 'active' state, mutants of AT1R will display a significantly reduced affinity with angiotensin II. However, if it has multiple functional states, different mutants of AT1R will display a broad range of affinities, some of which might even be higher than the affinity value of the wild-type AT1R-angiotensin II complex.

If a blocking agent (drug) is to be designed against AT1R, it should be such as to target the AT1R-angiotensin II complex with the highest affinity. Moreover, multiple derivatives of the drug should be tested to determine which one has the highest affinity against AT1R. Drug designing can also be done using computer simulations to select potential inhibitors before testing them.