VACM-1, a cullin gene family member, regulates cellular signaling

Maria Burnatowska-Hledin, P. Zhao, B. Capps, A. Poel, K. Parmelee, C. Mungall, A. Sharangpani, and L. Listenberger

Departments of Biology and Chemistry, Peale Science Center, Hope College, Holland, Michigan 49422-9000


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Vasopressin-activated Ca2+-mobilizing (VACM-1) receptor binds arginine vasopressin (AVP) but does not have amino acid sequence homology with the traditional AVP receptors. VACM-1, however, is homologous with a newly discovered cullin family of proteins that has been implicated in the regulation of cell cycle through the ubiquitin-mediated degradation of cyclin-dependent kinase inhibitors. Because cell cycle processes can be regulated by the transmembrane signal transduction systems, the effects of VACM-1 expression on the Ca2+ and cAMP-dependent signaling pathway were examined in a stable cell line expressing VACM-1 in VACM-1 transfected COS-1 cells and in cells cotransfected with VACM-1 and the adenylyl cyclase-linked V2 AVP receptor cDNAs. Expression of the VACM-1 gene reduced basal as well as forskolin- and AVP-stimulated cAMP production. In cells cotransfected with VACM-1 and the V2 receptor, the AVP- and forskolin-induced increases in adenylyl cyclase activity and cAMP production were inhibited. The inhibitory effect of VACM-1 on cAMP production could be reversed by pretreating cells with staurosporin, a protein kinase A (PKA) inhibitor, or by mutating S730A, the PKA-dependent phosphorylation site in the VACM-1 sequence. The protein kinase C specific inhibitor Gö-6983 further enhanced the inhibitory effect of VACM-1 on AVP-stimulated cAMP production. Finally, AVP stimulated D-myo-inositol 1,4,5-trisphosphate production both in the transiently transfected COS-1 cells and in the stable cell line expressing VACM-1, but not in the control COS-1 and Chinese hamster ovary cells. Our data demonstrate that VACM-1, the first mammalian cullin protein to be characterized, is involved in the regulation of signaling.

adenylyl cyclase; adenosine 3',5'-cyclic monophosphate; protein kinase A; protein kinase C; D-myo-inositol 1,4,5-trisphosphate


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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VASOPRESSIN-ACTIVATED CALCIUM-MOBILIZING (VACM-1) receptor is a 780-amino acid protein originally cloned from a rabbit medullary cDNA library (7). Expression of the cloned VACM-1 protein in COS-1 cells results in increased arginine vasopressin (AVP) binding (dissociation constant of about 2 nM) and in increased AVP-dependent mobilization of cytosolic-free Ca2+ (7). 125I-labeled AVP binding to membranes from COS-1 cells expressing VACM-1 is inhibited by the V1 antagonist [1(beta -mercapto-beta ,beta '-cyclopentamethylene propionic acid),2-(O-methyl)tyrosine]Arg8-vasopressin (MeAVP) and the V2 receptor agonist 1-desamino-8-D-arginine vasopressin (DDAVP), but not by a 10,000-fold excess of oxytocin or bradykinin. A small but significant binding is observed with 3H-labeled V1 antagonist [3H]d(CH2)Tyr5Me-[3H]AVP, but no significant binding of 3H-labeled V2 antagonist [phenylalanyl-3,4,5-3H]dCH2-Ile5-Phe-Ile-Asn-Cys-Pro-Arg-OH, which differs structurally from the DDAVP, can be detected (7). The immunohistochemistry and immunoprecipitation studies using polyclonal antibodies prepared against a synthetic peptide derived from the NH2-terminal sequence of VACM-1 indicate that the VACM-1 receptor is a cell membrane protein (7). Importantly, however, VACM-1 does not share any structural or sequence homology with the cloned V1 and V2 receptors (7). Consequently, we concluded that the VACM-1 receptor differs from the pharmacologically defined V1 and V2 receptors and may represent a novel receptor for AVP or a receptor for an as yet unidentified peptide similar in structure to AVP (7). The biological role of the VACM-1 receptor is yet to be defined.

Interestingly, the human homologue of the VACM-1 receptor, identified by Byrd and co-workers (8), is a highly conserved protein, differing in only seven amino acids from the rabbit VACM-1 protein. The human VACM-1 gene was localized on chromosome 11q22-23 to a region close to the gene for ataxia telangiectasia and was previously associated with a loss of heterozygosity in breast cancer tumor samples (8). Because of the proximity of the VACM-1 gene to this region, and its sequence homology with a recently identified gene family termed cullins, which in yeast and nematodes regulate cell cycle transitions (18, 20), Byrd et al. (8) suggested that VACM-1 may be a possible tumor suppressor gene.

To date, six cullin genes have been identified in Homo sapiens, five in Caenorhabditis elegans, and three in Saccharomyces cervisiae (18, 20). Their biological role is not fully understood and may be rather diverse. For example, in C. elegans, cul-1 is required for developmentally programmed transition from G1 to the G0 phase (18), whereas its yeast homologue, cDc53, targets phosphorylated G1 cyclins for degradation via the ubiquitin-dependent pathway (20). Hs-cul-2 has been implicated in the tumor-suppressor activity of the von Hippel-Lindau gene product (22), and the expression of Hs-cul-3 and Hs-cul-4 is increased in cultured colon cancer cells (13) and in primary breast cancer cells (10), respectively. The biological function of cul-5, which shares 96% sequence homology with VACM-1 (157/163 amino acids at their COOH terminus), remains unknown.

In an attempt to further characterize the VACM-1 receptor and to elucidate the significance of its link to both the V1 AVP receptor and to cullins, we have recently examined its tissue expression at the mRNA and at the protein level. Whereas Northern blot and RT-PCR analyses indicated that VACM-1 mRNA may be present in numerous tissues (5, 7, 8), the immunostaining of tissue sections with polyclonal antibodies prepared against synthetic peptides derived from the sequence of VACM-1 indicates that the VACM-1 receptor is expressed specifically in the vascular endothelial cells. In the kidney, it is also present in the medullary collecting tubule cells (5). Because those structures are well recognized for their role in controlling fluid homeostasis, we have speculated that VACM-1 may be involved in the regulation of vascular endothelial cells and the collecting tubule permeability (5).

The peptide hormone AVP is an effective vasoconstrictor, regulates cell permeability (14, 16), and is recognized as a growth factor that can affect cell proliferation and tissue regeneration (24, 28). These diverse effects of AVP involve either the cAMP- or the Ca2+-signaling pathways (6, 14, 16, 28). We were thus intrigued by the possibility that VACM-1 may be involved in the regulation of these signaling pathways. The work reported here shows that VACM-1, when expressed in vitro, significantly reduces AVP- and forskolin (FSK)-stimulated adenylyl cyclase activity and cAMP production and increases AVP-dependent D-myo-inositol 1,4,5-trisphosphate (IP3) production. The mammalian VACM-1 receptor, therefore, may represent a unique protein involved in the regulation of cell signaling.


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MATERIALS AND METHODS
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Materials. DMEM and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY). Myo-[3H]inositol and [32P]ATP were purchased from DuPont-NEN (Boston, MA). AVP was purchased from Peninsula Laboratories (Belmont, CA), and fura 2-AM was purchased from Molecular Probes (Junction City, OR). Staurosporin and Gö-6983 were purchased from Calbiochem (San Diego, CA).

Tissue culture. COS-1 cells were grown in DMEM supplemented with 10% FBS at 37°C under a water-saturated 7% CO2 atmosphere. Cells were subcultured for 24 h before transfection at a density of 6-8 × 105 cells per 100-mm plate. Cells were transfected with 5 µg VACM-1 cDNA per 100-mm culture dish using the diethylaminoethyl-dextran method. For the stable expression of VACM-1 cDNA in DHG44-CHO dihydrofolate reductase (dhfr-) cells, EMC3 vector, kindly donated by the Genetic Institute (Cambridge, MA), was used. VACM-1 cDNA was cut with unique Sal I/Xba I restriction enzymes from pSV-SPORT-1 vector and ligated into the Sal I/Xba I site in the polylinker region of EMC3 vector upstream from the dhfr gene allowing coamplification of the dhfr and VACM-1 cDNAs. Based on a single-point [3H]AVP binding assay and an increase in Ca2+ in response to AVP, we identified three clone lines expressing VACM-1 receptor. The cells were cloned by limited dilution, and "clone 1" was chosen for all subsequent studies.

cAMP measurements. Cells were incubated with various doses of effectors or vehicle (simplified saline solution, SSS) (5, 6) for 10 min. After the treatment, the reaction was stopped with ice-cold 12% TCA, and cells were frozen at -80°C. Cells were then centrifuged for 15-20 min, and the supernatant was separated for measurements of cAMP. cAMP production was analyzed either in duplicate or in triplicate using either a 125I-cAMP RIA (5, 6) or a nonradioactive cAMP kit from Amersham (Arlington Heights, IL).

Adenylyl cyclase activity. Adenylyl cyclase activity was assayed in quadruplicate as described by Birnbaumer and Yang (4). Briefly, cells grown and treated as described above were rinsed with Tris-buffered saline (10 mM Tris · HCl, 150 mM NaCl, pH 7.4) and incubated for 2 h in 50 ml of solution containing 0.1 mM [alpha -32P]ATP, 2 mM MgCl, 10 mM GTP, 1 mM EDTA, 1 mM cAMP, 2 mM methyl isobutyl xanthine, 50 mg/ml saponin, 20 mM creatinine phosphate, 20 mg/ml creatinine phosphokinase, 20 mg/ml myokinase, and 5 mM p-nitrophenyl phosphate. [3H]cAMP (10,000 cpm/well) was added for determination of cAMP recovery. The reaction was stopped by adding 40 mM ATP, 10 mM cAMP, and 1% SDS solution. The relative number of cells per well was determined by measuring the hydrolysis of p-nitrophenyl phosphate (A405). cAMP was isolated by chromatography on Dowex 50-XB and Alumina columns. Eluates were collected in scintillation vials and quantitated by scintillation counting.

IP3 measurement. IP3 was measured using the assay by Berridge (3) as described previously (6). Briefly, cells were incubated for 6 h with 15 µCi of myo-[3H]inositol at 37°C and exposed to either AVP (10-6 M) or vehicle (SSS) for 10 min. For some assays, 10 µg/ml of pertussis toxin was added to the incubation medium. After the 6-h incubation with pertussis toxin or vehicle, and after the treatment with AVP, the reaction was stopped with ice-cold 12% TCA, and cells were put on a shaker for 15 min to disrupt the membranes. Cells were then centrifuged for 15-20 min, and the supernatant was separated for measurements of inositol phosphates using AG-1X8 resin columns (Bio-Rad Laboratories). Inositol 1-phosphate was eluted with 200 mM ammonium formate/100 mM formic acid, D-myo-inositol 1,4-bisphosphate with 400 mM ammonium formate/100 mM formic acid, and IP3 with 1 M ammonium formate/100 mM formic acid. The pellet was resuspended in 0.5 ml of 1 mM KCl, 10 mM myo-inositol, and 0.5 ml of chloroform for total lipid measurements. The results were calculated as counts per minute of [3H]inositol phosphates/counts per minute of [3H]lipids and expressed as a percentage of the control.

Measurement of Ca2+ mobilization. Intracellular concentrations of free Ca2+ in COS-1 cells were measured spectrophotometrically with an AB2 dual-excitation spectrofluorometer (SLM-Aminco) using fura 2-AM as a Ca2+ indicator as described previously (7). However, to sufficiently load Chinese hamster ovary (CHO) and stably VACM-1 transfected CHO cells (clone 1) with fura 2-AM, cells were incubated with 4 µM fura 2-AM in the presence of 12.5 µM sulfinpyrazone, a potent inhibitor of organic anion transport, 1% FBS, and 50 µM sucrose at room temperature for 60 to 90 min (19). In the absence of sulfinpyrazone, we could not detect any responses to either AVP or ionomycin. Cells were washed two times with 5 ml of SSS, centrifuged, resuspended in 300 µl of SSS, and used in measurements of Ca2+ concentration.

Site-directed mutagenesis. Site-directed mutagenesis was performed using a QuickChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). The mutagenesis primers were synthesized by Sigma-Genosys (Woodland, TX), and the mutation site sequences were confirmed by sequencing performed by Sequi-net (Colorado State Univ., Ft. Collins, CO). The altered nucleotide sequences are underlined. S720A mutation primers were: forward, 5'GAG AAA GAA AAT TGC TAA TGC TCA ACT GCA GAC TG3', reverse: 5'CAG TCT GCA GTT GAG CAT TAG CAA TTT TCT TTC TC3'. The T325A mutation primers were: forward, 5'CCA AGA GCA CTT CTT TAA GCT TTG CTT CAA TCT CTT CAG AAG CAA GCT TTT TG3', reverse: 5'GCA AAA AGC TTG CTT CTG AAG AGA TTG AAG CAA AGC TTA AAG AAG TGC TCT TGG3'.

Statistical analysis. Data are expressed as means + one SE of the mean. As appropriate, either ANOVA or a paired Student's t-test was used for data analysis. Significance was set at P < 0.05.


    RESULTS
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We found that the expression of VACM-1 cDNA in COS-1 cells reduces basal cAMP production and inhibits isoproterenol (IP, 10-8 M)- and FSK (10-4 M)- induced increases in cAMP (Fig. 1A). These results were similar using either a 125I-RIA or a nonradioactive kit to measure cAMP production. Similarly, in clone 1 cells in stably expressing VACM-1, the basal and FSK-induced increase in cAMP production was attenuated compared with that in the control CHO cells (Fig. 1B). Finally, 10-6 M AVP induced a significant increase in cAMP production in CHO cells transfected with the V2 cDNA, but did not affect cAMP production in clone 1 cells transfected with the V2 cDNA (Fig. 1C).


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Fig. 1.   The effects of 10-6 M arginine vasopressin (AVP), 10-4 M forskolin (FSK), and 10-6 M isoproterenol (IP) on cAMP production in cells expressing vasopressin-activated Ca2+-mobilizing (VACM-1) receptor. A: control (cont) COS-1 and VACM-1 transfected COS-1 cells (n = 5, in triplicate; * P < 0.05 VACM-1 transfected group compared with the control COS-1 cells). B: the effects of AVP and FSK on cAMP production in Chinese hamster ovary (CHO) and clone 1 (Cln 1) cells expressing VACM-1 (n = 6, * P < 0.05 compared with control). C: the effects of AVP and FSK on cAMP production in CHO and clone 1 cells cotransfected with a vasopressin V2 receptor cDNA (n = 3, * P < 0.05 compared with control). Prot, protein.

To confirm the direct involvement of adenylyl cyclase in the VACM-1-dependent regulation of cAMP production, the enzyme activity was assessed directly. Our data, shown in Fig. 2A, indicate that a dose-dependent stimulation of adenylyl cyclase activity by FSK in CHO cells was largely absent in clone 1 cells. Only at 100 µM FSK was a small increase in adenylyl cyclase activity observed. Similarly, AVP increased adenylyl cyclase activity in CHO cells transfected with the V2 receptor, but not in clone 1 cells transfected with the V2 receptor (Fig. 2B). To confirm these VACM-1-dependent changes in adenylyl cyclase activity, the above experiments were repeated in COS-1 cells transiently transfected with VACM-1 cDNA, V2 cDNA, or VACM-1 and V2 cDNAs. As shown in Fig. 2C, the effects of FSK and AVP were significantly attenuated in cells expressing VACM-1 but not in mock transfected COS-1 cells.


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Fig. 2.   The effects of AVP and FSK on adenylyl cylase activity in cells expressing VACM-1 receptor. A: the effects of FSK on adenylyl cyclase activity in CHO and clone 1 cells cotransfected with a vasopressin V2 receptor cDNA (n = 3). B: the effects of AVP on adenylyl cyclase activity in CHO and clone 1 cells transfected with the V2 receptor cDNA (n = 3). C: the effects of AVP on adenylyl cyclase activity in COS-1 and VACM-1 transfected COS-1 cells. Repeated in quadruplicate, n = 3.

To examine a potential role of protein kinase A (PKA) and protein kinase C (PKC) in the VACM-1-dependent regulation of cAMP production, we tested the effects of their inhibitors, staurosporin and Gö-6983, respectively. Although staurosporin has been used in the micromolar range to inhibit both enzymes in vitro (15, 23), its established 50% inhibitory concentration (IC50) for PKC and PKA is 0.7 nM and 7 nM, respectively. Consequently, the effects of 0.7, 10, and 500 nM staurosporin were tested in this study. Our data indicate that 0.7 nM staurosporin had little effect on VACM-1-inhibited AVP-dependent cAMP production (Fig. 3A, group 3). The effect of 10 nM staurosporin was tested on two independent cell preparations. As previously, in the absence of staurosporin, AVP increased cAMP production by 275% in control cells transfected with the V2 cDNA and by 156% in the V2 and VACM-1 cDNA cotransfected cells (n = 2). In cells pretreated with 10 nM staurosporin, AVP increased cAMP production by 221% in control cells expressing the V2 receptor and by only 183% in cells cotransfected with the V2 and VACM-1 cDNAs (n = 2). In cells treated with 500 nM staurosporin, VACM-1 expression no longer attenuated the AVP-increased cAMP concentration (Fig. 3A, group 4). To ascertain the specificity of this action, the effect of 500 nM staurosporin on basal cAMP production was tested (n = 2). As before, in the absence of staurosporin, AVP increased cAMP concentration by 280% in the V2 transfected cells and by 175% in the V2 and VACM-1 cDNA transfected cells. In the same cell cultures pretreated with 500 nM staurosporin, AVP increased cAMP concentration by 300% in control cells transfected with the V2 receptor only and by 600% in the V2 and VACM-1 cDNA cotransfected cells. To confirm the specificity of the PKA-dependent action of VACM-1, the effects of AVP were examined in cells cotransfected with the V2 receptor and with VACM-1 cDNA that had the PKA-dependent phosphorylation sites S720 and T325 (5) mutated to alanine. These results (summarized in Fig. 3, groups 7 and 8, respectively) indicate that S730A but not T325A mutation of the PKA-dependent phosphorylation sites in VACM-1 cDNA completely reverses the attenuating effect of VACM-1 on AVP-dependent cAMP production. Finally, we examined the effect of the PKC-specific inhibitor Gö-6983 (IC50: for PKCalpha and PKCbeta , 7 nM; for PKCgamma , 10 nM; and for PKCzeta , 60 nM) on AVP-dependent cAMP production in the V2 and VACM-1 cDNA cotransfected cells. As shown in Fig. 3A, groups 5 and 7, both 7 nM and 700 nM Gö-6983 enhanced the inhibitory effect of VACM-1 on AVP-stimulated cAMP production.


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Fig. 3.   The effects of 10-6 M AVP (A) and 10-4 M FSK (B) on adenylyl cylase activity in control COS-1 cells expressing the V2 receptor, (group 1, n = 6); COS-1 cells expressing V2 and VACM-1 receptors (groups 2-6) and treated with 0.7 nM staurosporin (group 3, n = 4), 500 nM staurosporin (group 4, n = 4), 7 nM Gö-6983 (group 5, n = 4), 700 nM Gö-6983 (group 6, n = 3); COS-1 cells transfected with S730A mutated VACM-1 and V2 receptors, (group 7, n = 4); and COS-1 cells transfected with S730A mutated VACM-1 and V2 receptors (n = 4). * P < 0.05 vs. group 1; # P < 0.05 vs. group 2.

The effects of staurosporin, Gö-6983, and mutagenesis of PKA-specific phosphorylation sites in VACM-1 cDNA on FSK-dependent regulation of cAMP production in the V2 receptor cDNA transfected cells and in V2 and VACM-1 cDNA cotransfected cells are summarized in Fig. 3B. At 0.7 nM, staurosporin further enhanced the inhibitory effect of VACM-1 on cAMP levels (Fig. 3B, group 3 vs. group 2). At 10 nM staurosporin, the inhibitory effect of VACM-1 cDNA on FSK-induced increase in cAMP concentration was attenuated (n = 2, data not shown). Pretreatment of cells with 500 nM staurosporin reversed the inhibitory effect of VACM-1 protein expression on FSK-induced change in cAMP levels (group 4). Mutation of S720A or T325A in the VACM-1 sequence did not have a clear effect on the FSK-induced changes in cAMP production (Fig. 3B, groups 7 and 8) because these changes were no longer significantly different from either the control (group 1) or VACM-1 transfected cells (group 2). Finally, 7 nM Gö-6983 further enhanced the inhibitory effect of VACM-1 on FSK-induced changes in cAMP production (Fig. 3B, group 5). At 700 nM, Gö-6983 reversed the inhibitory effect of VACM-1 on FSK-dependent cAMP production (Fig. 3B, group 6).

To examine the mechanism by which AVP induces VACM-1-dependent changes in Ca2+ (5), the effects of AVP on IP3 production were measured in stably transfected CHO cells (clone 1) and in transiently transfected COS-1 cells. As shown in Fig. 4A, 10-6 M AVP treatment had no significant effect on IP3 production in the untransfected CHO cells (n = 5). The effect of AVP on IP3 production in the stable cell line (clone 1) was most pronounced at 1.5 min (n = 5, P < 0.05, compared with the time 0 or with the control CHO cells). A pretreatment of clone 1 cells with 10 µg/ml pertussis toxin for 6 h inhibited the AVP-induced increase in IP3 production (Fig. 4A, n = 5). The effect of AVP on IP3 production was associated with changes in the cytosolic-free Ca2+ concentration (Fig. 4A, inset). Similarly, AVP significantly increased IP3 production in COS-1 cells transfected with VACM-1 cDNA but not in sham-transfected COS-1 cells (Fig. 4B). This effect of AVP on IP3 production in cells transfected with VACM-1 was comparable to its well-documented effect in A7r5 and in 3T3 cell lines (data not shown).


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Fig. 4.   The effects of 10-6 M AVP on IP3 production in control cells and in cells expressing VACM-1 protein. A: CHO cells and clone cells were divided into 2 aliquots and were either sham treated or pretreated with pertussis toxin (PT; 10 µg/ml) for 6 h, at which time all cells were treated with 10-6 M AVP for 10 min. Repeated in triplicate, n = 4. * P < 0.05 when clone 1 cells () were compared with the PT-treated clone 1 cells (black-triangle) or with control CHO cells (). # P < 0.05 when AVP-treated clone 1 cells were compared with time 0 of the AVP treatment. Inset: an example of the effect of 1 µM AVP on Ca2+. B: the effects of AVP on IP3 production in COS-1 cells () and transiently VACM-1 transfected COS-1 cells (, means of 3 independent preparations performed in duplicate). * P < 0.05 compared with time 0 or with the untransfected control cells.


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ABSTRACT
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This study demonstrates that IP3 and cAMP-signaling pathways, affected by G protein-linked ligands that include AVP and by growth-regulating factors (2, 11, 13, 24, 28), are altered in cells that express VACM-1 protein. Both stable and transient expression of VACM-1 confers upon cells the ability to inhibit cAMP production (Fig. 1). The inhibitory effect of VACM-1 expression on cAMP production is associated with decreased adenylyl cyclase activity (Fig. 2). Further, in cells expressing VACM-1, AVP treatment increases IP3 production (Fig. 4) that may be responsible for the subsequent increase in Ca2+ (5). These opposite effects on the two signaling pathways indicate that the inhibitory action of VACM-1 on cAMP synthesis is specific and not due to a general downregulation of signaling pathways. The inhibitory effect of VACM-1 may be dependent on the levels of VACM-1 expressed (data not shown) and can be overcome by a high concentration of AVP (Fig. 2B). The observation that VACM-1 may also affect the isoproterenol-dependent cAMP production (Fig. 1A) suggests that these attenuating effects of VACM-1 protein may not be limited to the action of AVP. Whether the expression of VACM-1 affects other types of membrane receptors remains to be elucidated. Finally, the inhibitory action of VACM-1 expression on the FSK-dependent adenylyl cyclase activity and cAMP production suggests that this effect may even be independent of the ligand binding.

Although the cellular mechanism of the downregulating effect of VACM-1 protein on adenylyl cylcase activity is not clear, the ability of VACM-1 to attenuate the AVP and FSK-induced production of cAMP appears to depend on its phosphorylation status by PKA and PKC. Staurosporin, at doses that inhibit PKA activity, either attenuated or reversed the VACM-1-dependent decrease in cAMP production in response to AVP and to FSK. Although staurosporin's established IC50 for PKC and PKA is 0.7 nM and 7 nM, respectively, at high concentrations, it can be a general kinase inhibitor (15, 23). Consequently, its PKA specificity described here could be questioned. We therefore reexamined the responses to AVP and FSK in cells expressing the V2 receptor and cotransfected with VACM-1 cDNA with its two putative PKA phosphorylation sites, S730 and T325 (5), mutated to alanine. Elimination of the S730 but not the T325 PKA phosphorylation site of VACM-1 resulted in a complete inhibition of the VACM-1 activity, thus suggesting a direct effect of PKA on the VACM-1 receptor. Unlike staurosporin, PKC inhibitor Gö-6893, regardless of its concentration, further enhanced VACM-1-dependent downregulation of AVP-induced changes in cAMP concentration. However, the effect of the S730A mutation on the FSK-dependent regulation of cAMP is less clear. Although the inhibitory effect of VACM-1 in those experiments was attenuated, the differences were not statistically significant when compared with either the control cells or with cells cotransfected with the wild-type VACM-1 cDNA. Similarly, Gö-6893, at a concentration close to its IC50 for PKCalpha and PKCbeta (7 nM), enhanced the attenuating effect of VACM-1 on the FSK-induced changes in cAMP production. The attenuating effect of VACM-1 on the FSK-induced changes in cAMP, however, was reversed with high concentrations of the PKC inhibitor. Although the reasons for these differences are not clear, it is possible that at high doses, Gö-6893 also inhibits PKA activity. It is also possible that the sensitivity of the AVP- and FSK-regulated pathways to VACM-1 varies, or that only specific isoforms of adenylyl cyclase are regulated by VACM-1. So far, nine mammalian isoforms of the enzyme have been identified, cloned, and shown to differ in their responses to regulatory molecules and in their tissue specificity (17, 25, 26). Whereas all adenylyl cyclase subtypes can be stimulated by Gsalpha and by FSK, only some can be stimulated by Ca2+ calmodulin, and some are inhibited by Gi, Ca2+, and PKA. Some cyclases can also be regulated by the beta gamma -subunits of the G protein and by PKC (25, 26). For example, isoforms I, III, and VII can be stimulated by Ca2+ calmodulin and Gsalpha , isoforms IV and VII by Gsalpha and Gbeta gamma , and isoforms V and VI can be inhibited by Gialpha and Ca2+ (21, 25, 26). In the kidney, where VACM-1 expression is specific to the collecting tubule and to vascular endothelial cells, types IV, V, VI, VII, and IX of adenylyl cyclase have been identified (25). The isoforms of adenylyl cyclase expressed by either the CHO or the COS-1 cells used here have not yet been identified. Consequently, it remains to be elucidated whether the attenuating effect of VACM-1 expression on cAMP production is specific to a particular isoform of adenylyl cyclase or is limited by the type expressed in the system where it is studied.

Another level of complexity within the adenylyl cyclase signaling system is presented by the modulatory activity of the G proteins that can further differentiate the adenylyl cyclase-dependent signaling systems. For example, different combinations of the isoforms of the Gbeta gamma have an opposite effect on the same adenylyl cyclase isoforms (1). Whether these modulatory proteins are also involved in the action of VACM-1 described above is not apparent from our studies. Whereas the V2 receptor-dependent stimulation of adenylyl cyclase by AVP involves the activation of the Gsalpha , VACM-1 shares pharmacological characteristics with the V1 receptors (5) that are coupled to the Gq/11, and in some instances, to the Gi protein (29). Our previous work suggested that the effects of purine and pyrimidine nucleotides on binding of AVP to membranes from COS-1 cells transfected with VACM-1 cDNA were inconsistent with the typical selective effects of GDP and GTP on binding of ligands to G protein-linked receptors (5). However, the presence of a G protein-binding consensus sequence in VACM-1 (5, 21) and reports that PKC inhibits AVP-stimulated cAMP accumulation via a Gi-dependent mechanism (27) have led us to examine potential involvement of a pertussis toxin-sensitive G protein in the VACM-1 signaling. Our data suggest that pretreatment of cells expressing VACM-1 with pertussis toxin for 6 h inhibits IP3 production (Fig. 4) but does not appear to reverse the inhibitory effect of VACM-1 on the AVP- or FSK-regulated cAMP production (data not shown). Consequently, the possibility that the VACM-1-dependent action on adenylyl cyclase activity involves G protein remains to be determined.

Although VACM-1 has been identified as a vasopressin (AVP) binding receptor, it does not share any structure or sequence homology with the AVP receptors. Further, AVP-binding characteristics of VACM-1 protein and its tissue distribution do not match that of the V1 or the V2 AVP receptors (5, 7). Consequently, the relevance of its specific expression in the collecting tubule and in endothelial cells to the well-established biological effects of AVP in those structures remains unclear. Similarly unclear is the significance of its high sequence homology with recently identified cullins. Unlike cullins, which are either nuclear or cytosolic proteins (18, 22), VACM-1 expression localizes to the cell membrane (6, 7). Also, unlike cullins that appear to be rather ubiquitous (18), VACM-1 protein is expressed primarily in the vascular endothelial cells and in the renal collecting tubule cells (5). Finally, the function of cul-5, which shares highest sequence homology with VACM-1 (96% identity in the last 153/168 amino acids), has not been determined to date. Because the highest sequence homology between cullins and VACM-1 is observed in their COOH-terminal sequence, whereas the putative transmembrane region has been identified at the NH2-terminal of VACM-1 (amino acids 259-279), it is possible that VACM-1-related proteins lacking the transmembrane region may exist.

In summary, VACM-1, which is specifically expressed in the vascular endothelial cells and renal collecting tubule cells (5), when expressed in vitro, attenuates cAMP-dependent signal transduction. Further, the biological activity of VACM-1 may depend on its phosphorylation status maintained by PKA and PKC activities. Because VACM-1 shares high sequence homology with cullin proteins that regulate cell cycle, these data suggest an additional role for this largely uncharacterized multigene family.


    ACKNOWLEDGEMENTS

We thank Dr. Mariel Birnbaumer (Dept. of Anesthesiology, University of California at Los Angelos School of Medicine) for kindly providing the V2 cDNA and Dr. Chris Barney (Hope College) for helpful suggestions in preparing this manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-47199, Howard Hughes Medical Institute Grant 71196-528502, and by National Science Foundation-Research Experience for Undergraduates Grants CHE9322088 and DBI9322220.

Address for reprint requests and other correspondence: M. Burnatowska-Hledin, Dept. of Biology, Peale Science Center, Hope College, Holland, MI 49422-9000 (E-mail: hledin{at}hope.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 21 June 1999; accepted in final form 27 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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