A single mutation in helix 8 enhances the angiotensin II type 1a receptor transport and signaling

Abstract

The amphipathic helix 8 in the membrane-proximal C-terminus is a structurally conserved feature of class A seven transmembrane-spanning G protein-coupled receptors (GPCRs). Mutations of this helical motif often cause receptor misfolding, defective cell surface transport and dysfunction. Surprisingly, we demonstrated here that a single point mutation at Lys308 in helix 8 markedly enhanced the steady-state surface density of the angiotensin II type 1a receptor (AT1aR). Consistent with the enhanced cell surface expression, Lys308 mutation significantly augmented AT1aR-mediated mitogen-activated protein kinase ERK1/2 activation, inositol phosphate production, and vascular smooth muscle cell migration. This mutation also increased the overall expression of AT1aR without altering receptor degradation. More interestingly, Lys308 mutation abolished AT1aR interaction with β-COP, a component of COPI transport vesicles, and impaired AT1aR responsiveness to the inhibition of Rab6 GTPase involved in the Golgi-to-ER retrograde pathway. Furthermore, these functions of Lys308 were largely dependent on its positively charged property. These data reveal previously unappreciated functions of helix 8 and novel mechanisms governing the cell surface transport and function of AT1aR.

Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology

“…The list of AT 1 R interacting proteins has now been expanded to include filamin A, a cross-linking signal transducer (1055), tubulin (1258), Coatomer subunit ␤ (␤-COP) (1275), and GABA receptor-associated protein (GABARAP) (182). Filamin A and tubulin are associated with the cellular cytoskeleton, and the tubulin/AT 1 R interaction contributes to AT 1 R trafficking to the cell surface (1055,1258).…”

“…␤-COP is a component of Coat Protein I (COPI) transport vesicles involved in the transport between different Golgi stacks and transport from the Golgi to the ER. ␤-COP and AT 1 R interaction is dependent on Lys 308 in the cytoplasmic domain of AT 1 R and functions to repress AT 1 R forward trafficking as a Lys 308 mutation enhances AT 1 R cell surface expression (1275). GABARAP is involved in GABA A receptor trafficking through microtubule networks.…”

AT1 receptor signaling pathways in the cardiovascular system

“…AT1 receptor also directly binds to filamin A, an actin cross-linking protein, with agonist activation of the AT1 receptor promoting filamin phosphorylation, suggestive of a direct role of AT1 receptor in actin remodeling mediated by filamin (131). β-COP (Coatomer subunit β), a component of Coat Protein I (COPI) transport vesicles involved in the transport between different Golgi stacks and transport from the Golgi to the ER, interacts with AT1 receptor on Lys308 and regulates AT1 receptor export trafficking to the cell surface (132). Taken together, AT1 receptor binds to various interacting proteins through C-terminal domain and facilitates diverse signaling including AT1 receptor trafficking and cell surface expression (Table 1).…”

Mechanisms of the anterograde trafficking of GPCRs: Regulation of AT1R transport by interacting proteins and motifs

“…There are four positively charged residues at positions 307, 308, 310 and 311 in the C‐terminal membrane‐proximal region of AT1R (Figure ). This positive cluster was shown to be required for the high affinity binding of the receptor to the negatively charged lipids of the plasma membrane, to contain a nuclear localization signal which mediates AT1R translocation into the nucleus, and to influence the total synthesis of the receptor …”

“…This positive cluster was shown to be required for the high affinity binding of the receptor to the negatively charged lipids of the plasma membrane, [144][145][146] to contain a nuclear localization signal which mediates AT1R translocation into the nucleus, 147,148 and to influence the total synthesis of the receptor. 149 In GST fusion protein pull-down assays to search for interacting proteins using the C-terminus of α 2B -AR as bait, tubulin was identified as an interacting protein of α 2B -AR. 83 Further mutagenesis analysis of the C-terminus identified R437, R441 and R446 in the membraneproximal region responsible for tubulin interaction.…”