2004 Global Researcher Conference Proceeding
April 09 - 11, 2004
| Conference: | 2004 Global Researcher Conference |
|---|---|
| Title: | A defect in AQP2 phosphorylation at Ser256 in the Golgi apparatus might explain the molecular basis of the dominant form of NDI caused by the mutation E258K-AQP2 |
| Authors: | Procino, Giuseppe; Carmosino, Monica; Pinna, Lorenzo A.; Svelto, Maria; Valenti, Giovanna |
| Institutions: | University of Bari, Universita di Padova, Universita di Bari |
An autosomal dominant form of Nephrogenic Diabetes Insipidus (NDI) is caused by a mutation of the Glu-258 in the vasopressin-sensitive water channel Aquaporin 2 (AQP2). When expressed in oocytes and in renal cells, AQP2-E258K was retained in the Golgi region, suggesting that the phenotype is caused by an impaired routing of AQP2. This disease model provided insight for understanding the role of AQP2 phosphorylation in regulating AQP2 trafficking.
Aquaporin 2 (AQP2) phosphorylation at Ser-256 by PKA is a key signal for vasopressin-stimulated AQP2 insertion into the plasma membrane in renal cells. This study underscores the possible role of phosphorylation at Ser-256 in regulating AQP2 maturation. AQP2-transfected renal CD8 cells were incubated with Brefeldin A (BFA) to accumulate newly synthesized AQP2 in the ER and AQP2 flow from ER to the vesicular compartment was analyzed after BFA washout. We found that: 1) in the ER, AQP2 is weakly phosphorylated 2) the amount of phosphorylated AQP2 (p-AQP2) at Ser-256 increased significantly during transit in the Golgi, even in the presence of the PKA inhibitor H89 3) AQP2 transport from the Golgi to the vasopressin-regulated vesicular compartment occurred with a concomitant decrease in p-AQP2 at Ser-256. These results support the hypothesis that AQP2 transition in the Golgi apparatus is associated with a PKA-independent increase in AQP2 phosphorylation at Ser-256. Conversely, impaired constitutive phosphorylation in a Golgi-associated compartment occurring in cells expressing mutated S256A-AQP2 or E258K-AQP2, causes phosphorylation-defective AQP2 routing to lysosomes. This result might explain the molecular basis of the dominant form of NDI caused by the mutation E258K-AQP2, in which the phenotype is caused by an impaired routing of AQP2.
Phosphorylation is a process whereby a phosphate group is induced into an organic molecule. Phosphorylation of AQP2 occurs at a specific amino acid residue identified as Ser 256 and provides a necessary step in the transfer of AQP2 from the cell interior to the cell membrane. However, Valenti, et al., have discovered that AQP2 phosphorylation at Ser 256 may also be an important part of the AQP2’s maturation process. In its development to a mature AQP2 able to move from the cell interior, the AQP2 must move first through a section of the cell called the endoplasmic reticulum (ER) then through a section called the Golgi Apparatus. From there, the AQP2 moves into tiny sacs that take it to the cell membrane.
Using laboratory cell cultures, the research team discovered:
- AQP2 is weakly phosphorylated in the ER.
- The amount of phosphorylated AQP2 increased significantly as it travels through the Golgi Apparatus.
- The AQP2s movement from the Golgi to the transportation sacs that take them to the cell membrane is accompanied by a decrease in phosphorylation. This indicates that AQP2s’ movement through the Golgi is associated with an increase in phosphorylation at Ser 256 that is independent of the PKA increase in AQP2 phophorylation that is part of the arginine vasopressin (AVP) stimulated concentration process.
The researchers reason that the lack of AQP2 in the Gogli apparatus may be the molecular basis of the dominant form of NDI caused by the mutation of E258K–AQP2.