Phosphorylation events and the modulation of aquaporin 2 cell surface expression

Abstract

Purpose of review: This review highlights the role of phosphorylation in the trafficking and targeting of aquaporin 2. Current knowledge will be put into the context of modulating the cell surface expression of aquaporin 2 by vasopressin in renal epithelial cells, which is critical for regulation of urinary concentration and control of fluid and electrolyte homeostasis.

Recent findings: In addition to previously identified phosphorylation sites on aquaporin 2, new data have revealed three other serine residues in the C-terminus whose phosphorylation is altered by vasopressin. Several steps in aquaporin 2 recycling, including exocytosis and endocytosis, are coordinated by phosphorylation and dephosphorylation to regulate cell surface accumulation. Aquaporin 2 phosphorylation on serine 256 regulates aquaporin 2 association with proteins that are involved in trafficking, including hsc/hsp70 and myelin and lymphocyte-associated protein.

Summary: Aquaporin 2 trafficking is regulated by phosphorylation of serine 256 and other amino acid residues in its cytoplasmic domain. These events increase or decrease interaction of aquaporin 2 with key regulatory proteins to determine the cellular distribution and fate of aquaporin 2, both after vasopressin addition and under baseline conditions. Better understanding of these mechanisms may provide new therapeutic avenues for patients with X-linked nephrogenic diabetes insipidus, as well as providing basic cell biological information relevant to membrane trafficking processes in general.

Figure 1

Aquaporin 2 recycling, including endocytosis and both constitutive and regulated exocytosisConstitutive exocytosis occurs independently of aquaporin 2 (AQP2) phosphorylation and involves recycling of AQP2 through a trans-Golgi or recycling endosome compartment. AQP2 membrane accumulation can be increased simply by inhibiting clathrin-mediated endocytosis. The regulated pathway occurs upon vasopressin (VP) interaction with its basolateral receptor (V2R), which increases cAMP formation after Gas stimulation of the adenylyl cyclase (AC). Protein kinase A (PKA) activation results in AQP2 phosphorylation initially on residue S256. At some point, S261 is dephosphorylated and S264 and S269 phosphorylation is increased. AQP2 phosphorylation can also be increased by the cGMP/protein kinase G (PKG) pathways, upon increased activity of the soluble guanylyl cyclase (GC) by, for example, nitric oxide (NO). Extracellular hypertonicity activates the mitogen activated protein (MAP) kinase pathway and JNK, ERK and p38 activities are all required for AQP2 surface accumulation after an acute hypertonic shock. During exocytosis, AQP2 interacts with SNARE proteins and their regulatory proteins such as Munc 18-2, and these interactions may be regulated by phosphorylation. Once at the cell surface, phosphorylated AQP2 resides in endocytosis-resistant domains, and its interaction with hsc70, a protein required for clathrin-mediated endocytosis, is inhibited. AQP2 that is not phosphorylated at S256 interacts strongly with hsc70 and this may be one of several protein–protein interactions that leads to AQP2 accumulation in clathrin-coated pits, followed by endocytosis. The myeloid and lymphocyte-associated protein (MAL) also is involved in AQP2 endocytosis by an as yet unknown mechanism. Endocytosis of AQP2 is also facilitated by protein kinase C (PKC) activation (but possibly not by direct phosphorylation of AQP2), as well as by activation of dopamine (DA, D1) and prostaglandin receptors (EP₃, PGE₂). Finally, the actin cytoskeleton is centrally involved in AQP2 trafficking: actin depolymerization alone results in cell surface accumulation of AQP2.