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Agonist and antagonist interaction with AT1 subtype angiotensin II receptors

Thursday, 5 July, 2007 - 16:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculty: Science and Bio-engineering Sciences
Tam Le Minh
phd defence

The octapeptide angiotensin II is the main peptide hormone of the renin-angiotensin
system (RAS) and is known to be involved in the physiological and pathological
regulation of blood pressure and cardiovascular function by stimulating AT1
receptors. Therapeutic agents that block the production of angiotensin II or inhibit its
interaction with the receptor have been proven to be highly efficient in reducing the
pathological effects. The AT1 receptor blockers are widely and effectively used for the
treatment of hypertension and congestive heart failure. In this study, the molecular
interaction between AT1 receptors and their ligands as well as the pharmacological
characterization of two newly developed AT1 receptor blockers were investigated on
intact CHO-K1 cells transiently and stably transfected with human AT1 receptors.
Rather than the former notion of two receptor conformations, an active and an
inactive one, the present study argues in favour of multiple ligand-stabilized receptor

In the first part, agonist and antagonist interactions to AT1 receptor were compared
between the wild type and mutated human AT1 receptors by radioligand binding and
functional studies. This provides information about the structural requirements for
AT1 receptor binding and activation by angiotensin II and its peptide fragments.
Based on our findings, a multi-step model for AT1 receptor activation was proposed
in which the receptor activation by angiotensin II occurs in at least two steps
involving a pre-activated and a fully active state. According to this model, amino acid
residues at the N-terminal part of angiotensin II play a key role in the pre-activation
process, which is initiated by the breaking of constraining intramolecular interactions
within the receptor. Subsequently the C-terminal part of this hormone is necessary
to drive the receptor into a fully active state.

In the second part, we investigated the pharmacological properties of the recently
developed non-peptide antagonists, olmesartan and telmisartan, by studying their
interaction with the AT1 receptor stably expressed in CHO-K1 cells. Using well
characterized in vitro methods, we found that both olmesartan and telmisartan are
competitive antagonists and that they displayed high affinity, slow dissociation, and
a high degree of insurmountability for the AT1 receptor. Our studies provide detailed
information about the molecular action mechanism of non-peptide antagonists and,
most importantly, that their interaction with the receptor is also a multi-step
process. A “two-state, two-step” model was proposed, in which the initial antagonistreceptor
interaction yields a fast reversible complex (i.e. surmountable state) and
then subsequently a more stable, tight binding antagonist-AT1 receptor complex (i.e.
insurmountable state).

Finally, we found that the binding and functional properties of the human AT1
receptor differed when comparing stably and transiently transfected CHO-K1 cells.
This may result from differences in receptor reserve (which was exclusively found in
the transiently transfected cells) and/or the receptor maturation. It is therefore
pertinent to take these issues into consideration when characterizing ligand-AT1
receptor interaction using the transient receptor expression system.