Unconventional Homologous Internalization of the Angiotensin II Type-1 Receptor Induced by G-Protein–Independent Signals

Abstract

Abstract-Internalization of a G-protein-coupled receptor (GPCR) is essential to the desensitization, endocytosis, and signal transduction of the receptor. It has been the general view that conventional homologous internalization of a GPCR requires activation of the G-protein(s) coupled to the receptor. However, whether and how GPCR-mediated G-protein-independent signals trigger receptor internalization remains unknown, although G-protein-independent internalization has been reported. Here we show that an angiotensin II (Ang II) type-1 (AT 1 ) receptor mutant incapable of activating any G-protein still undergoes normal internalization. Substitution of Asp 125 with Ala and Arg 126 with Leu at the highly conserved DRY motif of the AT 1 receptor disabled the ability of the receptor to activate G-proteins, as shown by various Ang II binding studies, GDP-GTP exchange, and inositol phosphate production assays. Surprisingly, the mutant internalized normally in the presence of Ang II and transactivated the epidermal growth factor receptor (EGFR). Similar to the wild-type receptor, overexpression of a dominant-negative K220R mutant GRK2 diminished the internalization of D125A-R126L but not the transactivation of EGFR. These data indicate that G-protein-independent specific signals may also trigger homologous internalizations of the AT 1 receptor through ␤-arrestin-dependent and -independent pathways, suggesting a possible mechanism for G-protein-independent activation of G-protein-coupled receptor kinases (GRKs). This may represent a general mechanism for triggering GPCR internalization.

Mutagenesis of important amino acid reveals unconventional homologous internalization of β1-adrenergic receptor

“…The result demonstrated that mutation of negatively charged aspartic acid at position 104 to positively charged amino acid of lysine functionally inactivated the receptor by uncoupling with G protein. Earlier, other similar types of investigation has been reported in β 2 -adrenergic (Chung et al 1988), α 2A adrenergic (Wang et al 1991) and AT 1 (Feng et al 2005) receptor due to mutation at conserved aspartic acid. On the other hand, the mutant Asp104Ala showed the higher basal level of cAMP production without agonistinduced stimulation, which might be another possible reason for demonstrating this abnormal signaling characteristic of receptor.…”

Internalization of Constitutively Active N111G Mutant of AT1 Receptor Induced by Angiotensin II–Receptor Antagonists Candesartan, Losartan, and Telmisartan: Comparison With Valsartan

“…The present study demonstrated the correlation between endocytosis and signal transduction of AT 1 receptors due to its site directed mutagenesis. Homologous internalization of GPCRs is an active process that requires specific ligand binding, conformational changes of the receptor, and signal transduction initiated by the activated receptor (13). To determine the possible reason for the behavior of the N111G mutant of the AT 1 receptor in signaling transduction, the present study investigated internalization in terms of cell localization of the receptor protein.…”

Constitutively Active Mutant N111G of Angiotensin II Type 1 (AT1) Receptor Induces Homologous Internalization Through Mediation of AT1-Receptor Antagonist

“…Agonist binding to a GPCR induces conformational changes in the receptor, leading to activation of Gαβγ heterotrimers (10). One function of the activated Gproteins is to activate GPCR kinases (GRKs) that in turn phosphorylate the specific receptor for desensitization.…”