Analysis of functional domains of angiotensin II type 2 receptor involved in apoptosis

We previously demonstrated that the intracellular third loop (i3 loop) of a ngiotensin II type 2 receptor (AT(2)) plays a key role in mediating the bio logical functions of this receptor. To determine which residues are importa nt for AT, signaling, mutated receptors with serial deletions within the i3 loop were stably expressed in PC12 cells. Deletion of residues 240-244 wit hin the intermediate portion of the i3 loop resulted in a complete loss of AT(2)-mediated apoptosis, inhibition of extracellular signal-regulated kina ses (ERK), and SHP-1 activation. In contrast to well characterized heptahel ical receptors, the AT, functions were not affected by deletions of the ami no- or carboxyl-terminal portions of the i3 loop. Alanine substitutions fur ther demonstrated that lysine 240, asparagine 242, and serine 243 are key r esidues for AT(2)-induced apoptosis, ERK inhibition, and SHP-1 activation. To examine whether a functional link exists between activation of SHP-1 and apoptosis, we used a catalytically inactive SHP-1 mutant and demonstrated that preventing SHP-1 activation strongly attenuates AT(2)-induced ERK inhi bition and apoptosis. Our data demonstrate that the intermediate portion of the i3 loop is important for AT(2) function and that SHP-1 is a proximal e ffector of the AT(2) receptor that is implicated in the inhibition of ERKs and in the apoptotic effect of this receptor.