Direct AKAP-mediated protein-protein interactions as potential drug targets.

A-kinase-anchoring proteins (AKAPs) are a diverse family of about 50 scaffolding proteins. They are defined by the presence of a structurally conserved protein kinase A (PKA)-binding domain. AKAPs tether PKA and other signalling proteins such as further protein kinases, protein phosphatases and phosphodiesterases by direct protein-protein interactions to cellular compartments. Thus, AKAPs form the basis of signalling modules that integrate cellular signalling processes and limit these to defined sites. Disruption of AKAP functions by gene targeting, knockdown approaches and, in particular, pharmacological disruption of defined AKAP-dependent protein-protein interactions has revealed key roles of AKAPs in numerous processes, including the regulation of cardiac myocyte contractility and vasopressin-mediated water reabsorption in the kidney. Dysregulation of such processes causes diseases, including cardiovascular and renal disorders. In this review, we discuss AKAP functions elucidated by gene targeting and knockdown approaches, but mainly focus on studies utilizing peptides for disruption of direct AKAP-mediated protein-protein interactions. The latter studies point to direct AKAP-mediated protein-protein interactions as targets for novel drugs.

Peptides for disruption of PKA anchoring.

Adaptor or scaffolding proteins are at the basis of multiprotein complexes that spatially and temporally co-ordinate the propagation and integration of a broad range of cellular events. One class of scaffolding proteins are AKAPs (A-kinase-anchoring proteins). They sequester PKA (protein kinase A) and other signalling molecules including phosphodiesterases, other protein kinases and protein phosphatases to specific subcellular compartments. AKAP-dependent protein-protein interactions play a role in many physiologically relevant processes. For example, AKAP-PKA interactions are essential for the vasopressin-mediated water re-absorption in renal collecting duct principal cells or beta-adrenoceptor-induced increases in cardiac myocyte contractility. Here, we discuss recently developed peptide disruptors of AKAP-PKA interactions. Such peptides are valuable tools to study the relevance of PKA anchoring in cellular processes.

Compartmentalized cAMP signalling regulates vasopressin-mediated water reabsorption by controlling aquaporin-2.

The cAMP/PKA (protein kinase A) signalling pathway is activated by a plethora of stimuli. To facilitate the specificity of a cellular response, signal transduction complexes are formed and segregated to discrete sites (compartmentalization). cAMP/PKA signalling compartments are maintained by AKAPs (A-kinase anchoring proteins) which bind PKA and other signalling proteins, and by PDEs (phosphodiesterases). The latter hydrolyse cAMP and thus limit its diffusion and terminate PKA activity. An example of a cAMP-dependent process requiring compartmentalization of cAMP/PKA signals is arginine-vasopressin-regulated water reabsorption in renal principal cells. A detailed understanding of the protein interactions within a signal transduction complex offers the possibility to design agents influencing PKA binding to a specific AKAP, the targeting of an AKAP or the interactions of AKAPs with other signalling molecules. The ability to specifically modulate selected branches of a signal transduction pathway would greatly advance basic research, and may lead to new drugs suitable for the treatment of diseases caused by dysregulation of anchored PKA signalling (e.g. renal and cardiovascular diseases).

Osmolality and solute composition are strong regulators of AQP2 expression in renal principal cells.

The water permeability of the renal collecting duct is regulated by the insertion of aquaporin-2 (AQP2) into the apical plasma membrane of epithelial (principal) cells. Using primary cultured epithelial cells from the inner medulla of rat kidney (IMCD cells), we show that osmolality and solute composition are potent regulators of AQP2 mRNA and protein synthesis, as well as the classical cAMP-dependent pathway, but do not affect the arginine vasopressin-induced AQP2 shuttle. In the presence of the cAMP analog dibutyryl cAMP (DBcAMP, 500 microM), NaCl and sorbitol, but not urea, evoked a robust increase of AQP2 expression in IMCD cells, with NaCl being far more potent than sorbitol. cAMP-responsive element-binding protein phosphorylation increased with DBcAMP concentrations but was not altered by changes in osmolality. In the rat and human AQP2 promoter, we identified a putative tonicity-responsive element. We conclude that, in addition to the arginine vasopressin/cAMP-signaling cascade, a further pathway activated by elevated effective osmolality (tonicity) is crucial for the expression of AQP2 in IMCD cells, and we suggest that the effect is mediated via the tonicity-responsive element.

Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells.

First published August 8, 2001; 10.1152/ajprenal.00091.2001.-We have recently demonstrated that actin depolymerization is a prerequisite for cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) into the apical membrane in AQP2-transfected renal CD8 cells (29). The Rho family of small GTPases, including Cdc42, Rac, and Rho, regulates the actin cytoskeleton. In AQP2-transfected CD8 cells, inhibition of Rho GTPases with Clostridium difficile toxin B or with C. limosum C3 fusion toxin, as well as incubation with the Rho kinase inhibitor, Y-27632, caused actin depolymerization and translocation of AQP2 in the absence of the cAMP-elevating agent forskolin. Both forskolin and C3 fusion toxin-induced AQP2 translocation were associated with a similar increase in the osmotic water permeability coefficient. Expression of constitutively active RhoA induced formation of stress fibers and abolished AQP2 translocation in response to forskolin. Cytochalasin D induced both depolymerization of F-actin and AQP2 translocation, suggesting that depolymerization of F-actin is sufficient to induce AQP2 translocation. Together, these data indicate that Rho inhibits cAMP-dependent translocation of AQP2 into the apical membrane of renal principal cells by controlling the organization of the actin cytoskeleton.

Ht31: the first protein kinase A anchoring protein to integrate protein kinase A and Rho signaling.

In an attempt to isolate protein kinase A anchoring proteins (AKAPs) involved in vasopressin-mediated water reabsorbtion, the complete sequence of the human AKAP Ht31 was determined and a partial cDNA of its rat orthologue (Rt31) was cloned. The Ht31 cDNA includes the estrogen receptor cofactor Brx and the RhoA GDP/GTP exchange factor proto-lymphoid blast crisis (Lbc) sequences. The Ht31 gene was assigned to chromosome 15 (region q24-q25). It encodes Ht31 and the smaller splice variants Brx and proto-Lbc. A protein of the predicted size of Ht31 (309 kDa) was detected in human mammary carcinoma and HeLa cells. Anti-Ht31/Rt31 antibodies immunoprecipitated RhoA from primary cultured rat renal inner medullary collecting duct cells, indicating an interaction between the AKAP and RhoA in vivo. These results suggest that Ht31/Rt31 represent a new type of AKAP, containing both an anchoring and a catalytic domain, which appears to be capable of modulating the activity of an interacting partner. Ht31/Rt31 have the potential to integrate Rho and protein kinase A signaling pathways, and thus, are prime candidates to regulate vasopressin-mediated water reabsorbtion.

Role and identification of protein kinase A anchoring proteins in vasopressin-mediated aquaporin-2 translocation.

The antidiuretic hormone arginine vasopressin (AVP) regulates water reabsorption in renal principal cells by inducing a cAMP/protein kinase A-dependent translocation of water channels [aquaporin-2 (AQP2)] from intracellular vesicles into the apical cell membranes. Using primary cultured rat inner medullary collecting duct (IMCD) cells, it has been shown that AQP2 translocation in response to AVP stimulation occurs only if protein kinase A (PKA) is anchored to PKA anchoring proteins (AKAPs), which are present in various subcellular compartments. The identity of the AKAPs involved has not yet been elucidated. One potential candidate is a new splice variant of AKAP18, namely AKAP18 delta.

Cell volume kinetics of adherent epithelial cells measured by laser scanning reflection microscopy: determination of water permeability changes of renal principal cells.

The water channel aquaporin-2 (AQP2), a key component of the antidiuretic machinery in the kidney, is rapidly regulated by the antidiuretic hormone vasopressin. The hormone exerts its action by inducing a translocation of AQP2 from intracellular vesicles to the cell membrane. This step requires the elevation of intracellular cyclic AMP. We describe here a new method, laser scanning reflection microscopy (LSRM), suitable for determining cellular osmotic water permeability coefficient changes in primary cultured inner medullary collecting duct (IMCD) cells. The recording of vertical-reflection-mode x-z-scan section areas of unstained, living IMCD cells proved useful and valid for the investigation of osmotic water permeability changes. The time-dependent increases of reflection-mode x-z-scan section areas of swelling cells were fitted to a single-exponential equation. The analysis of the time constants of these processes indicates a twofold increase in osmotic water permeability of IMCD cells after treatment of the cells both with forskolin, a cyclic AMP-elevating agent, and with Clostridium difficile toxin B, an inhibitor of Rho proteins that leads to depolymerization of F-actin-containing stress fibers. This indicates that both agents lead to the functional insertion of AQP2 into the cell membrane. Thus, we have established a new functional assay for the study of the regulation of the water permeability at the cellular level.

An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells.

Vasopressin regulates water reabsorption in renal collecting duct principal cells by a cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane. In the present work primary cultured inner medullary collecting duct cells were used to study the role of the proteins of the Rho family in the translocation of AQP2. Clostridium difficile toxin B, which inhibits all members of the Rho family, Clostridium limosum C3 toxin, which inactivates only Rho, and the Rho kinase inhibitor, Y-27632, induced both depolymerization of actin stress fibers and AQP2 translocation in the absence of vasopressin. The data suggest an inhibitory role of Rho in this process, whereby constitutive membrane localization is prevented in resting cells. Expression of constitutively active RhoA induced formation of actin stress fibers and abolished AQP2 translocation in response to elevation of intracellular cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced both depolymerization of the F-actin cytoskeleton and AQP2 translocation, indicating that depolymerization of F-actin is sufficient to induce AQP2 translocation. Thus Rho is likely to control the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.

The mechanisms of aquaporin control in the renal collecting duct.

The antidiuretic hormone arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Central to its antidiuretic action in mammals is the exocytotic insertion of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the apical membrane of principal cells, an event initiated by an increase in cAMP and activation of protein kinase A. Water is then reabsorbed from the hypotonic urine of the collecting duct. The water channels aquaporin-3 (AQP3) and aquaporin-4 (AQP4), which are constitutively present in the basolateral membrane, allow the exit of water from the cell into the hypertonic interstitium. Withdrawal of the hormone leads to endocytotic retrieval of AQP2 from the cell membrane. The hormone-induced rapid redistribution between the interior of the cell and the cell membrane establishes the basis for the short term regulation of water permeability. In addition water channels (AQP2 and 3) of principal cells are regulated at the level of expression (long term regulation). This review summarizes the current knowledge on the molecular mechanisms underlying the short and long term regulation of water channels in principal cells. In the first part special emphasis is placed on the proteins involved in short term regulation of AQP2 (SNARE proteins, Rab proteins, cytoskeletal proteins, G proteins, protein kinase A anchoring proteins and endocytotic proteins). In the second part, physiological and pathophysiological stimuli determining the long term regulation are discussed.

Protein kinase A anchoring proteins are required for vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells.

The antidiuretic hormone arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells by inducing a cAMP-dependent translocation of water channels (aquaporin-2, AQP-2) from intracellular vesicles into the apical cell membranes. In subcellular fractions from primary cultured rat inner medullary collecting duct (IMCD) cells, enriched for intracellular AQP-2-bearing vesicles, catalytic protein kinase A (PKA) subunits and several protein kinase A anchoring proteins (AKAPs) were detected. In nonstimulated IMCD cells the majority of AQP-2 staining was detected intracellularly but became mainly localized within the cell membrane after stimulation with AVP or forskolin. Quantitative analysis revealed that preincubation of the cells with the synthetic peptide S-Ht31, which prevents the binding between AKAPs and regulatory subunits of PKA, strongly inhibited AQP-2 translocation in response to forskolin. Preincubation of the cells with the PKA inhibitor H89 prior to forskolin stimulation abolished AQP-2 translocation. In contrast to H89, S-Ht31 did not affect the catalytic activity of PKA. These data demonstrate that not only the activity of PKA, but also its tethering to subcellular compartments, are prerequisites for cAMP-dependent AQP-2 translocation.

Low O-acetylation of sialyl-Le(x) contributes to its overexpression in colon carcinoma metastases.

Two factors potentially determining the consistent overexpression of sialyl-Le(x) antigen in colon carcinoma and metastases were investigated: (i) the expression of the mucins MUC1 and MUC2, known to carry sialyl-Le(x), by Northern blotting; (ii) the extent of sialic acid O-acetylation, by Western blotting and HPLC. RNA and sialyl-Le(x)-positive mucins were purified from normal colonic mucosa (N), primary carcinomas (T) and their liver metastases (M). Northern blots showed that mRNA expression both of MUC1 and of MUC2 decreases during the progression of the disease, and is lowest in metastatic tissue. The expression of mucin-bound sialyl-Le(x) increased strongly from N to T and, to a lesser extent, to M. After alkali treatment of the mucins these differences disappeared, indicating that the total amount of mucin-bound sialyl-Le(x) is the same in the 3 types of tissues. The O-acetylation of mucin-bound sialyl-Le(x) gradually decreased from N to M. HPLC analysis showed that in N about 70%, in T 45% and in M only 20% of mucin-bound sialic acids are O-acetylated. Thus, the increase of sialyl-Le(x) detectable during colon-carcinoma progression is due to diminished O-acetylation and not to increased expression of mucin protein cores. The decrease of O-acetylation is therefore the primary chemical alteration contributing to colon carcinoma-associated overexpression of sialyl-Le(x).

MUC2 gene suppression in human colorectal carcinomas and their metastases: in vitro evidence of the modulatory role of DNA methylation.

The development of the majority of colorectal carcinomas is associated with a diminished expression of the intestinal mucin MUC2 in the tumor cells. The significance and the mechanism of this alteration are not yet known. We sought to determine the molecular basis of this tumor-associated change and to investigate the extent to which it might also relate to metastases. MUC2 gene expression was compared in normal (N), carcinomatous (T), and metastatic tissues (M) from nine patients by immunohistochemistry, in situ hybridization, and Northern blotting. Immunohistochemistry and in situ hybridization showed consistently lower amounts of the expressed protein and mRNA in T and in M than in N; quantitative analysis by Northern blotting confirmed that the differences between MUC2 mRNA expression between N, T, and M were significant, the expression in metastases being less than 5% of that in the normal colonic tissue. The influence of DNA methylation as a possible regulatory mechanism of MUC2 gene expression was tested after the 5' and 3'-regions flanking the first exon of MUC2 were recovered from a genomic DNA library and used as probes in Southern blot. The DNA was isolated from colon carcinoma cell lines expressing MUC2 strongly (LS174T) or moderately (T84) and from that which was nonexpressing (Colo 205), and it was digested with the methylation-sensitive enzyme HpaII. The Southern blot patterns indicated that the increased methylation in the promoter region was concomitant with the decrease of MUC2 mRNA expression. Methylation of the promoter region ligated into a reporter vector suppressed the expression of the luciferase reporter gene in the three investigated cell lines. Furthermore, the expression of MUC2 gene was enhanced by treating the MUC2-expressing colon carcinoma cells with 5-aza-2'-deoxycytidine, a methylation-inhibiting agent. To our knowledge this is the first report to show that: (a) MUC2 gene is strongly suppressed in liver and lymph node metastases of colorectal carcinomas, and (b) suppression of MUC2 gene in colon carcinoma cells in vitro is associated with methylation of the promoter region.

Fucosyltransferase III and sialyl-Le(x) expression correlate in cultured colon carcinoma cells but not in colon carcinoma tissue.

The potential contribution of fucosyltransferases to the overexpression of sialyl-Le(x) antigen was investigated in the colon carcinoma cell line HT-29 and in human colon carcinoma tissue. In HT-29 cells as well as in normal or malignant colonic tissues Fuc-TIII, Fuc-TIV, Fuc-TVI but not Fuc-TV nor Fuc-TVII were detectable after RT-PCR. Sodium butyrate treatment of HT-29 cells increased (to about 200%) and DMSO treatment decreased (to about 20%) the expression of sialyl-Le(x). This modulation of sialyl-Le(x) was concomitant with the analogous increase/decrease of mRNA of Fuc-TIII but not Fuc-TIV. Fuc-TVI was not detectable by Northern blotting in HT-29 cells. In six human colon carcinomas which exhibited strong overexpression of sialyl-Le(x), the expression of Fuc-TIII-mRNA was the same or lower than in the corresponding normal colonic tissue. Thus Fuc-TIII expression may be affecting the expression of the sialyl-Le(x) moiety in HT-29 cells but not in human colon carcinoma tissue.

Expression of MUC2-mucin in colorectal adenomas and carcinomas of different histological types.

The expression of mucin MUC2 was investigated in normal colonic tissue, in colonic adenomas and in carcinomas of the mucinous and non-mucinous type. The latter were subdivided into carcinomas originating from the adenoma-carcinoma sequence (ACS) and de novo (DN) carcinomas. The expression was assayed by immunohistochemistry with the monoclonal anti-MUC2 antibody CCP58 and by mRNA semiquantitation. MUC2 protein epitope CCP58 was strongly expressed in 21% of normal colonic tissues, in 40% of villous and in 48% of tubular adenomas. Mucinous carcinomas exhibited strong expression in 72%, ACS carcinomas in 21% and DN adenocarcinomas in none of the tumors investigated. Compared with the adjacent non-malignant tissue (transitional mucosa), CCP58 epitope expression in the tumor was higher in 74% of mucinous carcinomas, but equal or lower in 69% of ACS carcinomas and in 100% of de novo carcinomas. The alterations of MUC2 expression detected by immunohistochemistry in adenocarcinomas were confirmed on mRNA level. These data indicate that the MUC2 expression pattern is different in the 3 carcinoma types investigated. MUC2 over-expression occurs in the adenomatous tissue. It is always maintained in mucinous carcinomas, but frequently decreased in non-mucinous ACS carcinomas. DN carcinomas are most frequently associated with decreased expression of MUC2.

Altered glycosylation of the MUC-1 protein core contributes to the colon carcinoma-associated increase of mucin-bound sialyl-Lewis(x) expression.

The mucin carbohydrate epitope sialyl-Le(x), detected with the monoclonal antibody AM-3, is strongly overexpressed in > 90% of human colon carcinomas. We show here that in colon carcinoma one of the mucin cores bearing the sialyl-Le(x) group is MUC-1, whereas sialyl-Le(x) present in normal colon is not detectable on MUC-1. The amounts of MUC-1 core detectable with the monoclonal antibody BC3 in extracts of tumor tissue are 60-180% of those in normal tissue. Two other carbohydrate epitopes located on MUC-1 in mucins from normal and tumor tissue have also been characterized. In contrast to sialyl-Le(x), their expression on MUC-1 is variable and does not correlate with the malignant transformation of colonic mucosa. The transfer of the sialyl-Le(x) group onto the MUC-1 core contributes to the colon carcinoma-associated overexpression of the sialyl-Le(x) epitope.