The Gβγ‐subunit interacts directly with aquaporin‐2 (AQP2) and regulates its membrane targeting

Abstract

Body water homeostasis depends on the trafficking of intracellular vesicles containing AQP2 to the apical plasma membrane of collecting duct principal cells. This is regulated mainly by the peptide hormone vasopressin (VP). The VP type 2 receptor (V2R) couples to the Gs protein, consisting of subunits Gsα and the dimer Gβγ. Gβγ exerts its effects through mechanisms including altered intracellular signaling and direct protein‐protein interactions, and this functional diversity of Gβγ is due to the existence of 12 different γ‐subunits. We have previously shown that AQP2 membrane targeting does not depend exclusively on the canonical Gsα‐mediated signaling pathway involving cAMP. We also determined that the Gβγ‐inhibitor gallein decreased V2R agonist (dDAVP)‐mediated AQP2 membrane targeting in mouse cortical collecting duct (mpkCCD) cells. In the present studies, we investigated the functional role of Gβγ and specific γ‐subunits on AQP2 membrane targeting and urinary concentration. Anaesthetized Sprague‐Dawley rats were pre‐treated with either gallein (1 μM) or vehicle (DMSO 0.1%). After 20 min infusion of dDAVP (0.01nM) in both groups, gallein‐treated rats had significantly lower urine osmolality than vehicle treated rats (598 +/− 58 vs 820 +/− 72). Brattleboro (VP‐deficient) rats were treated in vivo with either vehicle (saline) or arginine‐VP (1ng i.v.) and 80 min later, rats were killed and kidneys were processed for immunohistochemistry. Subsequently, the tissue was assessed for a possible protein‐protein interaction between Gβγ and AQP2 using an antibody‐based proximal ligation assay, Duolink. In the inner medullary collecting ducts of arginine‐VP treated rats, there was a 1.8‐fold significant increase in fluorescent punctae (representing Gβγ‐AQP2 interactions) relative to control animals. RT‐PCR identified expression of γ5, γ7, γ10 and γ12 Gγ‐subunits in mpkCCD cells. Stable cell lines expressing shRNA targeting each of these γ‐subunits were generated utilizing lentiviral transduction particles, resulting in 44–98 % knockdown (KD) at the mRNA level. In γ5 KD, γ10 KD and γ12 KD cells, during control conditions or dDAVP stimulation, no changes were observed in the number of Gβγ‐AQP2 Duolink‐punctae. In contrast, γ7 KD cells had a marked reduction both during control conditions and dDAVP stimulation. AQP2 membrane targeting was assessed in all cell lines using a cell surface biotinylation approach. In γ7 KD and γ10 KD cells, AQP2 membrane abundance was decreased compared with control transduced cells during dDAVP stimulation. In conclusion, Gγ7 and Gγ10 are involved in AQP2 membrane targeting. Gγ7 may induce its effects by initiating a direct protein interaction between Gβγ and AQP2, whereas the mechanism for Gγ10 is unknown.Support or Funding InformationETBO is supported by the The Danish Council for Independent Research (DFF) Medical Sciences, a Young Elite Researcher grant through the DFF Sapere Aude program grant 12‐127490 and The Boserup Foundation. RAF is supported by the DFF and Novo Nordisk Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.