Identification of protein kinase A signalling molecules in renal collecting ducts

Body water homeostasis is maintained by the correct balance between water intake and water loss through urine, faeces, sweat and breath. It is known that elevated circulating levels of the antidiuretic hormone vasopressin decrease urine volume to prevent excessive water loss from the body. Vasopressin/cAMP/protein kinase A (PKA) signalling is the canonical pathway in renal collecting ducts for phosphorylating aquaporin‐2 (AQP2) water channels, which leads to the reabsorption of water from urine via AQP2. Although recent omics data have verified various downstream targets of PKA, crucial regulators that mediate PKA‐induced AQP2 phosphorylation remain unknown, mainly because vasopressin is usually used to activate PKA as a positive control. Vasopressin is extremely potent and phosphorylates various PKA substrates non‐specifically, making it difficult to narrow down the candidate mediators responsible for AQP2 phosphorylation. The intracellular localization of PKA is tightly regulated by its scaffold proteins, also known as A‐kinase anchoring proteins (AKAPs). Furthermore, each AKAP has a target domain that determines its intracellular localization, enabling the creation of a local PKA signalling network. Although vasopressin activates most PKAs independently of their intracellular localization, some chemical compounds preferentially act on PKAs localized on AQP2‐containing vesicles while simultaneously phosphorylating AQP2 and its surrounding PKA substrates. Immunoprecipitation with antibodies against phosphorylated PKA substrates followed by mass spectrometry analysis revealed that the PKA substrate in proximity to AQP2 was lipopolysaccharide‐responsive and beige‐like anchor (LRBA). Furthermore, Lrba knockout studies revealed that LRBA was required for vasopressin‐induced AQP2 phosphorylation.


Introduction
Aquaporin-2 (AQP2) water channels in renal collecting ducts are essential for water reabsorption from urine to maintain the correct balance between water intake and water loss through urine, faeces, sweat and breath.Notably, dysfunction of the urinary concentrating system causes nephrogenic diabetes insipidus (NDI), which is characterized clinically by polyuria, polydipsia and hypernatraemia.In cases of severe NDI, the urine volume reaches 10−20 L/day (Sands et al., 2006).In contrast, sustained secretion of the antidiuretic hormone vasopressin overactivates AQP2, resulting in impaired water excretion and hyponatraemia; for example, in cases of congestive heart failure and syndrome of inappropriate secretion of antidiuretic hormone (Ishikawa, 2015).Thus, the determination of the molecular mechanisms underlying AQP2 regulation is necessary for a clear understanding of the pathophysiology of water homeostasis.
The AQP2 gene was cloned in 1993 (Fushimi et al., 1993), and this cloning represented a giant step towards the elucidation of the molecular mechanisms of urinary concentration.It is known that vasopressin is released from the posterior pituitary gland in response to elevated plasma osmolality.Subsequently, it binds to vasopressin type 2 receptors (V2Rs) in renal collecting ducts, thereby inducing the interaction between β-arrestins and the C-terminus tail of V2Rs.The V2R is a class B G-protein-coupled receptor (GPCR) that has higher affinity for β-arrestins than that for class A receptors, such as β 2 receptors.Furthermore, stable binding of β-arrestins initiates prolonged internalization of V2Rs in endosomes (Feinstein et al., 2013).The vasopressin-V2R-β-arrestin complex promotes recruitment of heterotrimeric G-proteins within the endosome, thereby forming a larger multi-protein complex (Marshall, 2016;Thomsen et al., 2016).Following this, the activated Gαs subunit dissociates from Gβγ subunits, resulting in enhanced activity of adenylyl cyclase (Adcy), especially Adcy6, and sustained production of cAMP.It has been reported that renal collecting duct-specific knockout of Adcy6 reduces vasopressin-stimulated cAMP accumulation by 40% (Roos et al., 2012).
The second messenger cAMP triggers various biological intracellular processes.Additionally, it is essential for the activation of AQP2 to increase water reabsorption from urine in the renal collecting ducts.In a previous study, arginine vasopressin receptor 2 (AVPR2; V2R) knockout mice died within 1 week after birth because of their high urine output and severe dehydration (Yun et al., 2000).Notably, the maximum increase in intracellular cAMP levels in response to the V2R agonist [deamino-Cys1, d-Arg8]-vasopressin (dDAVP) decreased by 50% in AVPR2 +/− mice (Yun et al., 2000), suggesting that intracellular cAMP levels are extremely low in AVPR2 knockout mice.In contrast, the elevation of cAMP levels improves urine concentration; thus, cAMP inducers [such as phosphodiesterase (PDE) inhibitors] are classically considered the drug target for congenital NDI caused by loss-of-function mutations in V2Rs (Ando & Uchida, 2018).
Phosphodiesterase inhibitors reduce the degradation of cAMP to AMP and induce the accumulation of intracellular cAMP.In a previous study, tissue cAMP levels in the inner medullary collecting ducts from an NDI mouse model were restored using the PDE3 inhibitor cilostamide and the PDE4 inhibitor rolipram (Coffey et al., 1991).Furthermore, the PDE4 inhibitor roflumilast, which is approved by the US Food and Drug Administration for the treatment of chronic obstructive pulmonary disease, significantly increased AQP2 phosphorylation at S256, S264 and S269 and decreased phosphorylation at S261 in rat inner medullary collecting ducts (Umejiego et al., 2017).In our previous study, we examined the effects of rolipram in an autosomal dominant NDI mouse model with frameshift mutations in AQP2 (763−772 del) (Sohara et al., 2006).In heterozygous knock-in mice, the mutant AQP2 formed AQP2 tetramers with wild-type AQP2, which has a dominant-negative effect on the normal translocation of wild-type AQP2 to the apical plasma membrane.Rolipram increases cAMP levels in the renal papilla and then activates AQP2 tetramers, which are composed of wild-type AQP2 alone, thereby leading to a partial improvement in urine osmolality.Phosphodiesterase inhibitors are effective in vivo; however, their efficacies in NDI mouse models are limited, suggesting the presence of other cAMP-generating GPCRs.In addition to V2R, there are at least two Gαs-coupled receptors, a calcitonin-related receptor-like receptor and a prostaglandin E 2 EP4 receptor, in renal collecting duct principal cells (Mehta et al., 2022).Interestingly, EP4 agonists can produce cAMP directly in renal collecting ducts and increase the water permeability (Li et al., 2009).Therefore, the use of GPCR agonists to increase cAMP production is another treatment option for congenital NDI.
There are various signalling components downstream of the cAMP signalling pathways that collectively regulate AQP2 transcription, phosphorylation, trafficking and water reabsorption from urine.Notably, AQP2 phosphorylation is a crucial factor that determines the intracellular localization of AQP2 (Fig. 1).Vasopressin changes the phosphorylation levels of four sites in the COOH-terminal tail of AQP2, namely Ser256, Ser261, Ser264 and Ser269.In particular, Ser256 is considered the master regulator of AQP2 activity because AQP2-S256A (which mimics non-phosphorylated AQP2) is resistant to cAMP stimulation and remains in intracellular vesicles, whereas AQP2-S256D (which mimics constitutively phosphorylated AQP2) remains at the apical plasma membrane even in the absence of cAMP (Fushimi et al., 1997;Tamma et al., 2011).Thus, determining the ways in which AQP2-S256 is phosphorylated in the vasopressin/cAMP signalling pathway is essential to understand the molecular mechanisms of urinary concentration.
Protein kinase A is involved in AQP2 phosphorylation at S261 and S269 AQP2-S256 (RRQS 256 ) is located in the PKA phosphorylation consensus site RRXS/T.Protein kinase A is a tetramer, consisting of two regulatory (PKA R) and two catalytic (PKAc) subunits, in its inactive form.cAMP directly binds to the PKA R subunits and induces conformational changes in them, thereby facilitating the release of the PKAc subunits from the PKA R subunits (Hao et al., 2019).Furthermore, the PKAc subunits phosphorylate RRXS/T sequences and other targets with R in position −3 relative to the phosphorylation site, such as RKXS/T (Chen et al., 2011;Hennrich et al., 2013).
AQP2-S256 is considered a PKA substrate because previous kinase assay studies have revealed that PKA directly phosphorylates the S256 of an AQP2 C-terminal peptide (AA: 241−271) in the presence of ATP (Hoffert et al., 2008).However, the development of an antibody specific to AQP2-S256 unexpectedly revealed that the phosphorylation levels of S256 are constitutively high, regardless of cAMP stimulation, especially in F. Ando and others J Physiol 602.13 in vitro experiments performed using mouse cortical collecting duct (mpkCCD) and Madin-Darby canine kidney cell lines (Ando et al., 2016;Yui et al., 2017).Furthermore, Xie et al. (2010) used the native rat collecting duct in their study and reported that the phosphorylation levels of S256 are also constitutively high and unresponsive to vasopressin.Conversely, S256 needs to be phosphorylated for downstream sites (including S269) to undergo phosphorylation (Hoffert et al., 2008).An analysis of phospho-specific AQP2 antibodies revealed that cAMP induces the dephosphorylation of AQP2-S261 and phosphorylation of AQP2-S269 (Moeller et al., 2009;Tamma et al., 2011).Although the phosphorylation of S256 promotes AQP2 exocytosis, the phosphorylation of S269 hinders AQP2 endocytosis, thereby additively accumulating AQP2 at the apical plasma membrane (Hoffert et al., 2008;Moeller et al., 2010).Importantly, it is known that S269 is not directly phosphorylated by PKA (Hoffert et al., 2008), which indicates that PKA indirectly changes AQP2 phosphorylation at S261 and S269.
Moreover, kinases putatively responsible for AQP2-S261 and AQP2-S269 have been reported (Brown et al., 2008); for example, Rho kinase, which phosphorylates S269 independently of PKA (Ranieri et al., 2020).The presence of these kinases makes the determination of the exact role of PKA in the vasopressin/cAMP signalling pathway more difficult.Furthermore, RRXS/T motif is known to be phosphorylated by PKA and several other kinases, including protein kinase G (PKG) and protein kinase C (PKC) of the AGC kinase group and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) (Johnson et al., 2023;Sugiyama et al., 2019).Additionally, PKG, PKC and CaMKII are known to be involved in the regulation of AQP2 phosphorylation (Bouley et al., 2000;Bradford et al., 2014;Brown et al., 2008;Cheung et al., 2020).Indeed, in a previous study, AQP2-S256 was phosphorylated by vasopressin even in PKA-knockout mpkCCD cells, supporting the idea that at least one kinase apart from PKA can phosphorylate S256 (Datta et al., 2020).
Although H89 is commonly used as a PKA inhibitor, it simultaneously inhibits other basophilic serine/threonine kinases, such as PKC and PKG, owing to its low specificity (Bradford et al., 2014).In a similar manner, the PKA inhibitor 14−22 amide myristoylated (myr-PKI; a substrate-competitive inhibitor and a peptide with RRXA sequence) is not specific for PKA, because kinases that recognize RRXS/T sequences bind competitively to myr-PKI.Notably, CRISPR/Cas9 genome editing technology has highlighted the specific inhibition of PKA activity.Isobe et al. (2017) generated PKA-knockout mpkCCD cell lines by genetic deletion of both PKAc subunits (α and β).RNA sequencing and quantitative proteomics based on stable isotope labelling of amino acids in cell culture revealed the complete loss of AQP2 mRNA and protein expressions by the PKA knockout.AQP2 protein abundance is largely dependent on PKA in mpkCCD cells.Overexpression of AQP2 in PKA-knockout mpkCCD cells indicates that PKA is required for cAMP-induced AQP2 trafficking to the apical plasma membrane.Although the phosphorylation level of AQP2-S256 is constitutively high and unresponsive to PKA knockout or activation by forskolin, the knockout significantly impairs the dephosphorylation of AQP2-S261 and phosphorylation of AQP2-S269; this finding confirms the indirect effects of PKA on AQP2 phosphorylation at S261 and S269.
The protein kinases downstream of PKA that are involved in AQP2 dephosphorylation at S261 and phosphorylation at S269 remain unknown.Quantitative phosphoproteome analysis provided a complete picture of the vasopressin signalling pathway in mpkCCD cell lines.It revealed that 81% of the 313 vasopressin-dependent phosphorylation sites are mediated by PKA and its six downstream protein kinases, namely Sik2, Cdk18, Camkk2, Prkd2, Mapk3 and Mylk (Datta et al., 2021).Furthermore, apart from kinases, protein phosphatases contribute to AQP2 phosphorylation in the vasopressin signalling pathway.It has been reported that PP2C inhibitors, rather than PP1, PP2A or PP2B inhibitors, hinder vasopressin-induced S261 dephosphorylation (Cheung et al., 2017(Cheung et al., , 2020)).In order to identify target kinases and phosphatases that regulate AQP2-S261 and AQP2-S269 directly, it is necessary to conduct knockout studies of these signalling molecules.Previous phosphoproteomics studies that used myosin light chain kinase (MLCK) and PKA-knockout mpkCCD cells revealed a significant overlap between MLCK and PKA signalling networks (Isobe et al., 2020).However, MLCK knockout did not exert any strong effect on AQP2 phosphorylation, which enabled us to narrow down candidate kinases and phosphatases responsible for AQP2 phosphorylation.

A-kinase anchoring proteins facilitate PKA-dependent AQP2 phosphorylation
Protein kinase A is recognized as a ubiquitously expressed protein kinase, and its intracellular localization and activity are strictly regulated by AKAPs, which are its scaffold proteins.There are 43 AKAP genes, and >70 functionally distinct AKAPs have been identified (Kapiloff et al., 2014).Each AKAP serves as a molecular platform that binds to PKA and its substrates, facilitating the phosphorylation of PKA substrates in a specific manner (Ando, 2021).Moreover, AKAPs bind to signalling molecules involved in the cAMP signalling pathway, such as PDEs, other kinases, phosphatases or other scaffolding proteins, thereby generating compartmentalized PKA signalling networks (Torres-Quesada et al., 2017).The fine-tuned balance between cAMP production by GPCR-dependent adenylyl cyclase and cAMP degradation by PDEs collectively limits its diffusion from AKAPs and activates PKA at the appropriate location and time (Johnstone et al., 2018).
Multi-omics data have indicated that ∼20 AKAPs are expressed in renal collecting ducts (Hara et al., 2022;Lee et al., 2015;Limbutara et al., 2020;Yang et al., 2015;Yu et al., 2009).Moreover, it has been reported that the AKAP220-PKA complex is co-localized with AQP2 in the cytosol of inner medullary collecting ducts.Forskolin-induced phosphorylation of overexpressed AQP2 in COS cells is enhanced by AKAP220 co-expression (Okutsu et al., 2008).However, AKAP220-PKA interaction is not essential for AQP2 phosphorylation in vivo.The phosphorylation status of AQP2 is unchanged in AKAP220-knockout mice (Whiting et al., 2016).AKAP220 knockout disrupts actin-barrier polymerization independently of PKA, allowing for the accumulation of AQP2 at the apical plasma membrane and reducing the urine-diluting capacity during overhydration.In addition, AKAP18δ is located on AQP2-containing vesicles.Co-immunoprecipitation assay with AKAP18δ antibody revealed that AQP2, PKA and PDE4D exist in the same vesicular fraction (Stefan et al., 2007).In response to vasopressin, the AKAP complex translocates to the apical plasma membrane, where PKA activates PDE4D to degrade cAMP and terminate cAMP-dependent AQP2 activation.A negative feedback loop is established through AKAP18δ, maintaining low cAMP levels in the vicinity of AQP2.AKAP18δ might contribute to PKA-induced AQP2 phosphorylation in vivo; however, AKAP18-knockout mice do not have an obvious phenotype of polyuria and polydipsia (Jones et al., 2012).Similar to AKAPs, STUB1 (an E3 ubiquitin ligase) organizes a signalosome consisting of PKA and AQP2 (Dema et al., 2020); however, PKA (which is anchored to STUB1) is not responsible for AQP2 phosphorylation.STUB1 knockdown does not affect the phosphorylation status of AQP2.
Although PKA is one of the major kinases in the vasopressin signalling pathway, the AKAP responsible for mediating PKA and AQP2 phosphorylation in vivo remains unknown.Moreover, to the best of our knowledge, there are no mouse models of AKAP knockout that exhibit polyuria.Studies on genetic polymorphisms, mutations and knockout have reported that defects in AKAP function cause various pathological cellular responses in cardiovascular disease, neurological disorders, cancers, obesity and osteoporosis (Suryavanshi et al., 2018).These diseases are generally caused by malfunctions of specific AKAP complexes and not by those of the global cAMP/PKA signalling pathway (Tröger et al., 2012).In order to identify the AKAP responsible for AQP2 phosphorylation in vivo, it is necessary to understand the local PKA signalling in the urinary concentration system.

Dissociation of the AKAP-PKA interaction phosphorylates AQP2
The development of strategies for treating congenital NDI caused by V2R mutations is a strong foundation for seeking to understand the regulatory mechanisms of PKA-induced AQP2 phosphorylation.In theory, the direct activation of PKA via bypassing of defective V2Rs can lead to AQP2 phosphorylation without vasopressin (Ando, 2021;Ando & Uchida, 2018).The dissociation of the interaction between AKAPs and PKA is a potential approach for the direct activation of PKA/AQP2 signalling.The peptide Ht31 is the most widely used AKAP-PKA disruptor that has a PKA-anchoring domain of a human thyroid AKAP (Carr et al., 1992).Ht31 and AKAPs bind competitively to type II regulatory subunits of PKA; thus, Ht31 dissociates AKAP-PKA interactions.Ht31 typically suppresses local PKA activity by releasing PKA from the AKAP-PKA-PKA substrate complex, whereas it activates PKA in baby hamster kidney cells (Ma et al., 2011).Moreover, in mpkCCD cell lines, PKA is activated initially by Ht31; subsequently, it induces the dephosphorylation of AQP2-S261 and phosphorylation of AQP2-S269 (Ando et al., 2018).
The proximity of PKA to PKA substrates is not always advantageous in terms of increasing the phosphorylation levels of PKA substrates.The PKA R subunit has four isoforms, namely RIα, RIβ, RIIα and RIIβ, and AKAP1 is known to be a dual-specific AKAP that binds to the PKA RI and PKA RII subunits.AKAP1 has a higher affinity for PKA RII subunits than for PKA RI subunits (Banky et al., 1998).In our model, PKA R subunits and mouse AKAP1 (mAKAP1) were overexpressed in HEK293T cells, and it was verified that mAKAP1 binds preferentially to PKA RII subunits (Fig. 2; Hara et al., 2022).Interestingly, the baseline phosphorylation levels of RRXS within AKAP1 were significantly decreased by overexpression of the PKA RIIα and PKA RIIβ subunits.In contrast, the overexpression of PKA RIIα and PKA RIIβ subunits did not affect forskolin-induced PKA activation or phosphorylation of mAKAP1.Based on the above-mentioned findings, it can be concluded that the range of action of AKAP1-anchored PKA was limited by AKAP1-PKA interaction in resting conditions, which inhibited basal AKAP1 phosphorylation.In such cases, Ht31 might increase the PKA activity and phosphorylate PKA substrates.
The effects of in vivo administration of Ht31 are limited owing to the low membrane permeability and short half-life of Ht31.The most promising drug targets are low-molecular-weight compounds that inhibit protein-protein interactions (Vukićević et al., 2016).Notably, Christian et al. (2011) discovered FMP-API-1 and its derivatives; for example, FMP-API-1/27, which binds to the allosteric site of PKA R subunits and dissociates AKAP-PKA interactions.Similar to Ht31, FMP-API-1 activates PKA without elevating intracellular cAMP levels, dephosphorylates AQP2-S261 and phosphorylates AQP2-S269 in mpkCCD cells (Ando et al., 2018).Furthermore, FMP-API-1 increases water permeability in isolated renal collecting ducts from mouse kidneys to the same extent as vasopressin.Although FMP-API-1 is ineffective in vivo because of its low potency, FMP-API-1/27, which strongly inhibits AKAP-PKA interactions compared with FMP-API-1 (Christian et al., 2011), phosphorylates AQP2 at S256 and S269 in mouse kidneys to the same extent as vasopressin (Ando et al., 2018).Thus, FMP-API-1/27 improved the urine concentrating ability in a V2R-inhibited NDI mouse model.
AKAP-PKA disruptors activate AQP2 without vasopressin in vivo; however, it is challenging to identify the target AKAP-PKA interaction dissociated by FMP-API-1/27.Thus, new screening methods are needed to detect AKAPs localized in AQP2-containing vesicles.

Lipopolysaccharide-responsive and beige-like anchor is the essential AKAP for urinary concentration
A-kinase anchoring proteins serve as subcellular address tags.Each AKAP has a targeting domain and is localized in a subcellular compartment (e.g.intracellular vesicles, nucleus or mitochondria), where it binds to PKA signalling molecules (Luconi et al., 2011;Torres-Quesada et al., 2017).Vasopressin activates almost all PKA signalling molecules non-specifically, regardless of the cellular compartment.Quantitative phosphoproteomic analysis revealed that dDAVP increased phosphorylation of various PKA substrates involved in vesicle trafficking, mRNA processing/translation, cytoskeleton regulation, cell-cell adhesion, proteolysis and mitochondrial function (Isobe et al., 2017).Moreover, an analysis of phospho-PKA substrate antibody, which recognizes phosphorylated RRXS/T sequences comprehensively, revealed that many PKA substrates are phosphorylated after the administration of dDAVP in mpkCCD cells (indicated by the red arrowheads in Fig. 3A).The potent effects of vasopressin are suitable as a positive control for PKA activation, but it is disadvantageous for uncovering the AKAPs necessary for AQP2 phosphorylation.
Activation of PKA by several compounds is an efficient screening strategy for addressing the above-mentioned problem (Hara et al., 2022).In comparison to vasopressin, FMP-API-1/27 phosphorylates a lower number of PKA substrates in mpkCCD cells (indicated by the blue arrowheads in Fig. 3A).Additionally, flavonoids, which increase cAMP levels through PDE inhibition, exhibit different phosphorylation patterns for PKA substrates.Some compounds simultaneously induce AQP2 phosphorylation at S269 by activating PKA localized in AQP2-containing vesicles (Fig. 3B).We found that the phosphorylation levels of PKA substrates (indicated by white dashed line in Fig. 3A) were almost perfectly correlated with those of AQP2-S269.Immunoprecipitation with phosphorylated PKA (pPKA) substrate antibody followed by mass spectrometry analysis revealed that the PKA substrate was lipopolysaccharide-responsive and beige-like anchor (LRBA) (Fig. 3C), which is an AKAP that is significantly homologous to DAKAP550 and which contains two putative PKA-binding sites (Wang et al., 2001).A co-immunoprecipitation assay of Ramos B-cell lymphoma cells revealed that endogenous LRBA interacts with PKA RIIβ, and this interaction is inhibited by Ht31 (Moreno-Corona et al., 2020).
The immunoprecipitation method with pPKA substrate antibody has several advantages.First, this antibody can detect pPKA substrates with high protein expression levels.It is known that western blotting does not detect proteins with low expression levels, which are less likely to be involved in AQP2 phosphorylation.Second, in the immunoprecipitation assay, proteins in proximity to AQP2 are extracted because their phosphorylation statuses are strongly correlated.In fact, LRBA and AQP2 are co-localized on the same vesicles (Chou et al., 2018;Hara et al., 2022).Third, the immunoprecipitation assay strategy is applicable to extra-renal cell lines and tissues for similar purposes.We used forskolin and pPKA substrate antibody and then discovered a previously unidentified PKA substrate (ZNF185) in human umbilical vein endothelial cells (Suzuki et al., 2023).A co-immunoprecipitation assay revealed that PKA RIIα interacts with the C-terminal fragment (amino acids 501−689) of ZNF185.In contrast, PKA RIIα does not interact with a ZNF185 deletion mutant lacking amino acids 534−605 (ZNF185-PKA).The dissociation between ZNF185-PKA and PKA led to impairment in PKA-induced phosphorylation of ZNF185-PKA after forskolin treatment.In endothelial cells, cAMP/PKA signalling changes actin structures F. Ando and others J Physiol 602.13 dynamically to enhance the stability of cell-cell junctions.ZNF185 mediates PKA-induced endothelial barrier stabilization.Finally, in the immunoprecipitation assay, PKA-interacting proteins can be extracted.The pPKA substrate antibody recognizes phosphorylated RRXS sites of the PKA-interacting proteins LRBA and ZNF185.Approximately 70% of AKAPs have RRXS/T sequences and can be immunoprecipitated by pPKA substrate antibody (Hara et al., 2022).Thus, the combined use of several compounds (e.g.cAMP inducers and PKA activators) and a pPKA substrate antibody is a cost-effective method of identifying molecules directly linked to the mechanisms of urinary concentration.
Lipopolysaccharide-responsive and beige-like anchor is the first known AKAP responsible for AQP2 phosphorylation in vivo.Lrba knockout impairs AQP2 phosphorylation at S256 and S269, AQP2 trafficking to the apical plasma membrane and urine concentration in response to vasopressin.Therefore, Lrba-knockout mice lose body weight rapidly when they are dehydrated.Moreover, LRBA contributes to the recycling of vesicular CTLA-4 receptor, a checkpoint immune molecule in regulatory T cells.It might induce local PKA signalling to regulate plasma membrane expression levels of constitutively recycled proteins.However, further detailed investigations of the mechanism of the LRBA-mediated vesicular trafficking system are needed to validate our results.

Conclusion
Vasopressin/cAMP/PKA signalling is the canonical pathway for AQP2 phosphorylation.Advancements in the analytical techniques of molecular biology have enabled the verification of the roles of PKA in the regulation of AQP2.Recent omics data have provided a complete picture of the PKA signalling pathway in renal collecting ducts.Narrowing down the candidate kinases, phosphatases and mediators responsible for AQP2 phosphorylation is crucial for a clear understanding of the pathophysiological mechanisms of urinary concentration.Combined use of several compounds and pPKA substrate antibody is a new strategy for the identification of key regulators in the vasopressin signalling pathway.