Lateral mobility of the phospholipase C-activating vasopressin V1-type receptor in A7r5 smooth muscle cells: a comparison with the adenylate cyclase-coupled V2-receptor.

The present work examines lateral mobility of the vasopressin V1-type receptor, representing the first determination of lateral mobility of a hormone receptor coupled to phospholipase C activation. The V1-receptor of A7r5 smooth muscle cells was characterized for [Arg8] vasopressin (AVP) binding properties and affinity for the fluorescent vasopressin analogue 1-deamino[8-lysine (N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP). TR-LVP was biologically active in A7r5 cells, inducing inositol 1,4,5-trisphosphate turnover in similar fashion to AVP. TR-LVP was used to specifically label the V1-receptor of living A7r5 cells, and lateral mobility of the V1-receptor was measured using the technique of fluorescence microphotolysis. The apparent lateral diffusion coefficient (D) at 37 degrees C was 5.1 x 10(-10) cm2/s, falling to 2.9 x 10(-10) cm2/s at 13 degrees C. These D values are higher than comparable values for the adenylate cyclase-activating vasopressin V2-receptor of LLC-PK1 renal epithelial cells analysed with the same fluorescent ligand. In contrast to the V2-receptor, no marked temperature dependence was observed for the V1-receptor mobile fraction (f). From 37 degrees C to 13 degrees C, f was relatively low (between 0.4 and 0.5) consistent with V1-receptor immobilization through internalization, which is rapid even at room temperature in A7r5 cells. These differences between V1- and V2-receptor lateral mobility are discussed in terms of the implications for their respective signal transduction systems.     

N-glycosylation plays a role in biosynthesis and internalization of the adenylate cyclase stimulating vasopressin V2-receptor of LLC-PK1 renal epithelial cells: an effect of concanavalin A on binding and expression.

The role of N-glycosylation in the function and biosynthesis of the vasopressin V2-receptor in LLC-PK1 renal epithelial cells was examined using various lectins and inhibitors operating at different steps of the glycosidic pathway. Tunicamycin, which blocks all N-glycosylation, and castanospermine, which inhibits glycosidase I and hence blocks formation of high-mannose-type N-glycosylated intermediates, resembled one another in affecting V2-receptor biosynthesis and internalization in a concentration-dependent manner. In contrast, swainsonine, an inhibitor of mannosidase II and hence of complex-type oligosaccharide formation, had no effect. Interestingly, the alpha-D-mannose/alpha-D-glucose-specific lectin concanavalin A, (Con A), in contrast to the beta-D-galactose-specific lectin ricin, had a marked effect on the V2-receptor in LLC-PK1 cells, increasing both receptor numbers up to twofold in vivo and specific [3H]AVP binding up to 50% in vitro in a concentration-dependent manner. The concentrations inducing half-maximal response were about 0.2 and 20 micrograms/ml for the in vivo and in vitro responses, respectively, implying distinct effects on V2-expression and ligand binding. That the in vitro effect on binding was due to a direct effect on the V2-receptor could be shown by the lack of a Con A effect on [3H]AVP binding in membranes prepared from LLC-PK1 cells down-regulated for the V2-receptor or from cells of the LLC-PK1 V2-receptor deficient mutant M18. All results were consistent with a functional role for N-glycosylation of the V2-receptor in LLC-PK1 cells.     

A novel LLC-PK1 renal epithelial cell mutant impaired in in vivo down-regulation of cAMP-mediated hormonal response.

A novel "cAMP-resistant" variant of LLC-PK1 renal epithelial cells which is impaired in in vivo down-regulation of response following hormonal stimulation of adenylate cyclase (AC) is described. Compared to parental cells, the BIB27 mutant exhibited markedly higher in vivo activation of cAMP-dependent protein kinase (cAMP-PK) in response to the hormones salmon calcitonin (SCT) or [Arg8]-vasopressin (AVP) or the AC activator forskolin. The activation of cAMP-PK subsequent to agonist stimulation also persisted much longer in the mutant than in LLC-PK1 cells, although the cAMP-PK of BIB27 cells was normal in terms of both absolute levels and regulation by cAMP in vitro. Intracellular cAMP accumulation was also much higher in BIB27 than in LLC-PK1 cells following agonist stimulation. Production of cAMP could be detected in BIB27 cells even 12 h after treatment with AVP or SCT, whereas cAMP production in LLC-PK1 had returned to basal within 1 and 8 h, respectively. High levels of free cAMP-PK catalytic (C) subunit in BIB27 persisted even 12 h after hormone addition, meaning that the higher cAMP production in BIB27 did not result in the normal down-regulation of cAMP-PK C subunit levels. In vitro AC activity in BIB27 cell homogenates could be stimulated by hormones or receptor-independent agonists, but to a lesser extent than in LLC-PK1 cell homogenates. The SCT and AVP concentrations promoting half-maximal AC activation in BIB27 cells were about 10- and 3-fold higher than parental, respectively. BIB27 accordingly appeared to possess a mutation in AC responsible for the impairment of both in vitro response to agonists and the normal in vivo down-regulation processes following hormonal stimulation.     

Codominant expression of a mutation affecting the cAMP-dependent protein kinase catalytic subunit in somatic cell hybrids of LLC-PK1 cells

The LLC-PK1 mutant cell lines FIB4 and FIB6 are affected in the catalytic (C) subunit of cAMP-dependent protein kinase (cAMP-PK) such that they possess less than 10% parental activity. However, by Western blot analysis they were shown to possess normal levels of C subunit protein. Somatic cell hybrids were derived between mutant and LLC-PK1 cells, and examined for complementation of the cAMP-PK lesion. Codominant expression of mutant and normal alleles was observed, in that somatic cell hybrids between FIB4 and LLC-PK1, and between FIB6 and LLC-PK1 cells, exhibited cAMP-PK activity 60-75% that of LLC-PK1 cells, intermediate between mutant and normal parental cell lines. The cAMP-PK of the FIB6 x LLC-PK1 and FIB4 x LLC-PK1 hybrids was examined by ion exchange chromatography. In contrast to the FIB6 and FIB4 mutants which lack an active Type I cAMP-PK, the hybrids retained levels of active Type I cAMP-PK greater than 30% that of LLC-PK1, concomitant with the retention of catalytic activity. It was concluded that the loss of Type I kinase in the FIB6 and FIB4 mutants is most likely a consequence of the lesion in the cAMP-PK C subunit. All somatic cell hybrids examined showed levels of cAMP-PK C subunit (as determined by Western blot analysis), and in vivo regulation of cAMP-PK activation (in response to hormonal or nonreceptor-mediated stimulation of adenylate cyclase), completely comparable to those of the parental LLC-PK1 cells. Hence, no aberrant regulation of either cAMP-PK subunit levels or cAMP-PK activities was evident in the somatic cells hybrids. All data were consistent with the hypothesis that FIB4 and FIB6 contain a structural mutation affecting the cAMP-PK catalytic subunit that is expressed phenotypically in the presence of the normal allele.     

Biotinyl analogues of vasopressin as biologically active probes for vasopressin receptor expression in cultured cells

Biotinyl analogues of [Arg8]vasopressin were synthesized with the biotinyl moiety at position 4. This involved the substitution of 2, 4-diaminobutyric acid (Dab) for Gln4 in [1-deamino-Arg8]vasopressin to give the parent peptide des-[Dab4,Arg8]vasopressin. Two biotinyl analogues with different spacers between the side chain of Dab4 and the biotinyl residue were then prepared and characterized in detail. The analogues retained high binding affinities for the V2-receptor in both bovine kidney membranes and LLC-PK1 renal epithelial cells and for the V1-receptor in rat liver membranes. Both analogues were as potent as [Arg8] vasopressin in stimulating the cAMP-dependent protein kinase and the production of urokinase-type plasminogen activator in LLC-PK1 cells, with concentration dependence consistent with receptor binding affinities. Avidin or streptavidin did not appear to reduce receptor binding or biological activity of the biotinyl analogues. The use of the biotinylated vasopressin analogue des-[Dab-(biotinylamido)hexanoyl4, Arg8]vasopressin together with fluorescein-labeled streptavidin as a fluorescent probe for the V2-receptor in LLC-PK1 cells demonstrated the following: 1) Specific binding of the biotinyl analogue shown by quantitative single-cell fluorescence measurements using the technique of fluorescence microphotolysis; 2) the V2-receptor visualized by fluorescence microscopy; and 3) the expression of the V2-receptor detected by flow cytometry.     

Dependence of urokinase-type-plasminogen-activator induction on cyclic AMP-dependent protein kinase activation in LLC-PK1 cells.

The activation of cyclic AMP-dependent protein kinase (cAMP-PK) in vivo was studied in LLC-PK1 pig kidney cells and the mutant cell lines M18 and FIB5, which have total levels of cAMP-PK catalytic-subunit and regulatory-subunit activities comparable with those of parental cells. The extent of cAMP-PK activation (release of active catalytic subunit from the holoenzyme) was directly correlated with the cellular cyclic AMP concentration in LLC-PK1 cells. In LLC-PK1 cells, as well as in the mutants M18 and FIB5, the extent of the induction of urokinase-type plasminogen activator (uPA) by the cyclic AMP-mediated effectors calcitonin, vasopressin and forskolin was directly correlated with the levels of activated catalytic subunit. The 'receptorless' mutant M18, which is impaired in calcitonin- and vasopressin-receptor function, did not show any activation of cAMP-PK or uPA production in response to either hormone, whereas cAMP-PK and uPA responses to forskolin were about 35% higher than in parental cells. Analysis of the FIB5-cell line revealed a lesion affecting the regulation of adenylate cyclase activity, whereby basal and stimulated (both receptor- and non-receptor-mediated) adenylate cyclase levels were less than 36% of those in parental cells. The activation of cAMP-PK in response to cyclic AMP effectors was similarly reduced, and uPA induction was concomitantly lower than that in parental cells. The results demonstrate the dependence of uPA induction by cyclic AMP effectors on dissociation of the cAMP-PK holoenzyme, implying the importance of activated free cAMP-PK catalytic subunit in this process. Thus it is concluded that the mutations in the cellular cyclic AMP-generating apparatus of the M18 and FIB5 cell lines impair uPA induction by preventing cAMP-PK activation.     

Pathway of urokinase-type plasminogen activator induction in the T47D and LLC-PK1 cell lines

Induction of urokinase-type plasminogen activator (uPA) in response to either reagents activating cAMP-dependent protein kinase (cAMP-PK) or the calcium ion phospholipid-dependent kinase (C-kinase) was compared in the LLC-PK1 and T47D cell lines. The two cell lines exhibited quantitatively different responses to calcitonin, to the phosphodiesterase inhibitor isobutylmethylxanthine, and to the adenylate cyclase activator forskolin. Both showed activation of cAMP-PK in response to all these reagents, with T47D cells displaying a greater extent of activation. T47D cells, however, failed to produce uPA in response to calcitonin, forskolin, or the cAMP analog 8-bromo-cAMP, whereas LLC-PK1 cells produced high levels of uPA in response to all these agents. Both cell lines responded to phorbol esters in terms of uPA induction, though to differing extents. Phorbol myristate acetate (PMA) was shown conclusively not to activate cAMP-PK in either cell line, even at concentrations 10-fold higher than those promoting maximal uPA induction. It was concluded that phorbol ester-mediated induction of uPA does not involve cAMP or cAMP-PK activation. These results are discussed in relation to proposed models concerning the role of cAMP-PK in uPA induction.     

Complementation between LLC-PK1 mutants affected in polypeptide hormone-receptor function

The mutant LLC-PK1 cell lines FIB6 and FIB5/N4 were examined for responsiveness to the polypeptide hormones calcitonin and vasopressin. Both mutants exhibited little or no activation of adenylate cyclase or cAMP-dependent protein kinase (cAMP-PK) in response to calcitonin, but responded to vasopressin. Analysis of calcitonin receptor function demonstrated that both mutants bound less than 9 fmol 125I-labeled salmon calcitonin/mg cellular protein, which was about 1% of parental activity (642 fmol calcitonin bound/mg). Concomitant with reduced calcitonin binding, both mutants exhibited increased vasopressin binding (greater than 272 fmol [[3H]Arg]vasopressin bound/mg) compared to parental (166 fmol bound/mg). The concentration of vasopressin for half-maximal stimulation of adenylate cyclase in both mutants was comparable to that for LLC-PK1 cells (40 pM) and hence the increased binding activity was concluded to be due to increased numbers of functional vasopressin receptors in the mutants. Somatic cell hybrids formed between each mutant and LLC-PK1 cells exhibited normal hormone binding and activation of cAMP-PK in response to both vasopressin and calcitonin. The mutations affecting receptor function in FIB6 and FIB5/N4 were accordingly concluded to be recessive. Somatic cell hybrids between FIB6 and FIB5/N4 showed no complementation of the mutant phenotype, indicating that both cell lines were affected in the same gene. In contrast, somatic cell hybrids between FIB5/N4 and the 'receptorless' mutant M18 (which lacks functional calcitonin and vasopressin receptors) exhibited approximately the same responsiveness to vasopressin and to calcitonin as LLC-PK1. Complementation between two different mutations affecting polypeptide receptor function was thus observed. The results are discussed in terms of a proposed common pathway for processing of calcitonin and vasopressin receptors.     

The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature.

The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and beta-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37 degrees C: the mobile fraction (f) was approximately 0.9, and the apparent lateral diffusion coefficient (D) approximately 3.0 X 10(-10) cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10 degrees C f was reduced to approximately 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional role of the NPxxY motif in internalization of the type 2 vasopressin receptor in LLC-PK1 cells

Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, we expressed FLAG-tagged wild-type V2R and a Y325F mutant V2R in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr³²⁵ with Phe³²⁵ prevented ligand-induced internalization of V2R determined by [³H]AVP binding and immunofluorescence but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells or for adenylyl cyclase-mediated signal transduction. polarized cell culture; tyrosine motif; µ1b adaptor motif; protein traffic     

Okadaic acid induction of the urokinase-type plasminogen activator gene occurs independently of cAMP-dependent protein kinase and protein kinase C and is sensitive to protein synthesis inhibition.

Urokinase-type plasminogen activator (uPA) gene expression in LLC-PK1 cells is induced by activation of cAMP-dependent protein kinase (cAMP-PK) or protein kinase C (PK-C). To determine whether protein phosphatases can also modulate uPA gene expression, we tested okadaic acid, a potent specific inhibitor of protein phosphatases 1 and 2A, in the presence and absence of cAMP-PK and PK-C activators. Okadaic acid by itself induced uPA mRNA accumulation. This induction was strongly attenuated by the inhibition of protein synthesis. In contrast, the inhibition of protein synthesis enhanced induction by 8-bromo-cAMP and only delayed induction by 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, down-regulation of PK-C by chronic treatment with TPA did not abrogate the okadaic acid-dependent induction. These results provide evidence for a novel signal transduction pathway leading to gene regulation that involves protein phosphorylation but is independent of both cAMP-PK and PK-C.     

Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function.

A novel mutant of the LLC-PK1 renal epithelial cell line, VPR1, was isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine] vasopressin. The VPR1 mutant cell line possessed less than 5% parental V2 receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK1 cells (wild type), VPR1 cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo cAMP production, or urokinase-type plasminogen activator induction. The responses of VPR1 cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5'-[beta, gamma-imino] triphosphate, 8-bromo adenosine-3',5'-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK1 and VPR1 and analyzed for the dominance/recessiveness of the VPR1 mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V2 receptor in VPR1 cells is recessive. The VPR1 cell line may thus have application as a recipient for the expression of the V2 receptor gene using DNA-transfer.     

Vasopressin V2-receptor mobile fraction and ligand-dependent adenylate cyclase activity are directly correlated in LLC-PK1 renal epithelial cells

The role of hormone receptor lateral mobility in signal transduction was studied using a cellular system in which the receptor mobile fraction could be reversibly modulated to largely varying extents. The G-protein-coupled vasopressin V2-type receptor was labeled in LLC-PK1 renal epithelial cells using a fluorescent analogue of vasopressin, and receptor lateral mobility measured using fluorescence microphotolysis (fluorescence photobleaching recovery). The receptor mobile fraction (f) was approximately 0.9 at 37 degrees C and less than 0.1 at 10 degrees C, in accordance with previous studies. When cells were incubated for 1 h at 4 degrees C without hormone, and then warmed up to 37 degrees C and labeled with the vasopressin analogue, f increased from approximately 0.4 to 0.8 over approximately 1 h. The apparent lateral diffusion coefficient was not markedly affected by temperature pretreatment. Studies with radiolabeled vasopressin indicated that temperature pretreatment influenced neither receptor number nor binding/internalization kinetics. F-actin staining revealed that temperature change resulted in reversible changes of cytoskeletal structure. The maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin, but not to forskolin (receptor-independent agonist), was also markedly influenced by preincubation of cells at 4 degrees C, thus paralleling the effects of temperature preincubation on f. A linear correlation between f and maximal cAMP production was observed, suggesting that the receptor mobile fraction is a key parameter in hormone signal transduction in vivo. We conclude that mobile receptors are required to activate G-proteins, and discuss the implications of this for signal transduction mechanisms.     

Ammonium chloride affects receptor number and lateral mobility of the vasopressin V2-type receptor in the plasma membrane of LLC-PK1 renal epithelial cells: role of the cytoskeleton

The acidotropic agent ammonium chloride (NH4Cl) not only affects receptor metabolism by inhibiting lysosomal acidification, but can also affect the targeting of proteins to specific membranes in polarized cells, possibly through effects mediated by the cytoskeleton. The present study examines the effects of NH4Cl and perturbers of cytoskeleton structure on vasopressin V2 receptor expression in LLC-PK1 renal epithelial cells. Surprisingly, long-term pretreatment of cells with NH4Cl or short-term treatment with the actin perturber cytochalasin B resulted in an up to 70% increase in specific Arg-8-vasopressin binding compared to control cells, which was independent of the presence of NH4Cl in the binding test, and apparently the result of increased V2 receptor expression. Perturbers of microtubules such as colchicine and vinblastine had no such effect. A rhodamine-labeled analog of vasopressin was used to fluorescently label the V2 receptor of LLC-PK1 cells, and microscopic measurements of membrane-localized fluorescence confirmed the increased V2 receptor expression in the basal plasma membrane subsequent to NH4Cl pretreatment. Lateral mobility of the V2 receptor was measured in living cells using the technique of microphotolysis (photobleaching). The fraction of mobile receptors was 0.2 in cells pretreated with NH4Cl, markedly reduced compared to that of 0.9 in untreated cells. The apparent lateral diffusion coefficient D was about 3 x 10(-10) cm2/s in both pretreated and untreated cells. Results for fluorescence labeling of the actin cytoskeleton indicate that NH4Cl pretreatment of LLC-PK1 cells results in perturbation of microfilament structure. All results imply that the cytoskeleton plays a central role in V2 receptor expression and lateral mobility.     

Downregulation of the vasopressin type 2 receptor after vasopressin-induced internalization: involvement of a lysosomal degradation pathway

Vasopressin (VP) increases urinary concentration by signaling through the vasopressin receptor (V2R) in collecting duct principal cells. After downregulation, V2R reappears at the cell surface via an unusually slow (several hours) "recycling" pathway. To examine this pathway, we expressed V2R-green fluorescent protein (GFP) in LLC-PK1a cells. V2R-GFP showed characteristics similar to those of wild-type V2R, including high affinity for VP and adenylyl cyclase stimulation. V2R-GFP was located mainly in the plasma membrane in unstimulated cells, but it colocalized with the lysosomal marker Lysotracker after VP-induced internalization. Western blot analysis of V2R-GFP showed a broad 57- to 68-kDa band and a doublet at 46 and 52 kDa before VP treatment. After 4-h VP exposure, the 57- to 68-kDa band lost 50% of its intensity, whereas the lower 46-kDa band increased by 200%. The lysosomal inhibitor chloroquine abolished this VP effect, whereas lactacystin, a proteasome inhibitor, had no effect. Incubating cells at 20°C to block trafficking from the trans-Golgi network reduced V2R membrane fluorescence, and a perinuclear patch developed. Cycloheximide reduced the intensity of this patch, showing that newly synthesized V2R-GFP contributed significantly to its appearance. Cycloheximide also inhibited the reappearance of cell surface V2R after downregulation. We conclude that after downregulation, V2R-GFP is delivered to lysosomes and degraded. Reappearance of V2R at the cell surface depends on new protein synthesis, partially explaining the long time lag needed to fully reestablish V2R at the cell surface after downregulation. This degradative pathway may be an adaptive response to allow receptor-ligand association in the hypertonic and acidic environment of the renal medulla. lysosome; trafficking; vasopressin receptor in LLC-PK1 cells     

Internalization of V2-vasopressin receptors in LLC-PK1-cells: evidence for receptor-mediated endocytosis.

The mechanism of internalization of the vasopressin-receptor (V2-subtype) of LLC-PK1-cells, a pig renal tubular cell line, is unknown. We studied internalization utilizing a novel, highly specific vasopressin analogue ((125I)-[8-p(OH)-phenylpropionyl]-LVP, 2000 Ci/mmol). Scatchard analysis performed with membranes of LLC-PK1-cells revealed a Kd of 0.8 +/- 0.2 nM and a Bmax of 366 +/- 41 fmol/mg of protein. Degradation of the ligand was excluded by RP-HPLC-analysis. Internalization was proven by the acid-wash technique, quantitative light-microscopic autoradiography and electron microscopy. The ligand was internalized in a time- and temperature-dependent manner. At 4 degrees C, no uptake was found; at 22 degrees C, after 30 min of incubation, more than 50% of the radioligand was found inside the cell. Electron microscopy demonstrated that plasma-membrane bound vasopressin receptors are internalized by receptor-mediated endocytosis via coated pits.     

alpha 2B-adrenergic receptor activates MAPK via a pathway involving arachidonic acid metabolism, matrix metalloproteinases, and epidermal growth factor receptor transactivation

We have investigated the mechanisms whereby alpha(2B)-adrenergic receptor (alpha(2B)-AR) promotes MAPK activation in a clone of the renal tubular cell line, LLC-PK1, transfected with the rat nonglycosylated alpha(2)-AR gene. Treatment of LLC-PK1-alpha(2B) with UK14304 or dexmedetomidine caused arachidonic acid (AA) release and ERK2 phosphorylation. AA release was abolished by prior treatment of the cells with pertussis toxin, quinacrine, or methyl arachidonyl fluorophosphonate but not by the addition of the MEK inhibitor U0126. The effects of alpha(2)-agonists on MAPK phosphorylation were mimicked by cell exposure to exogenous AA. On the other hand, quinacrine abolished the effects of UK14304, but not of AA, suggesting that AA released through PLA2 is responsible for MAPK activation by alpha(2B)-AR. The effects of alpha(2)-agonists or AA were PKC-independent and were attenuated by indomethacin and nordihydroguaiaretic acid. Treatment with batimastat, CRM 197, or tyrphostin AG1478 suppressed MAPK phosphorylation promoted by alpha(2)-agonist or AA. Furthermore, conditioned culture medium from UK14304-treated LLC-PK1-alpha(2B) induced MAPK phosphorylation in wild-type LLC-PK1. Based on these data, we propose a model whereby activation of MAPK by alpha(2B)-AR is mediated through stimulation of PLA2, AA release, generation of AA derivatives, activation of matrix metalloproteinases, release of heparin-binding EGF-like growth factor, transactivation of epidermal growth factor receptor, and recruitment of Shc. Whether this pathway is particular to alpha(2B)-AR and LLC-PK1 or whether it can be extended to other cell types and/or other G-protein-coupled receptors remains to be established.     

LLC-PK1 cell mutants in cAMP metabolism respond normally to phorbol esters

Mutants of the pig kidney cell line, LLC-PK1, affected in cAMP metabolism, were examined for cAMP-dependent protein kinase (cAMP-PK) activity and for cAMP-mediated induction of urokinase-type plasminogen activator (uPA). The FIB4 and FIB6 mutant cell lines possessed about 10% parental levels of cAMP-PK activity and concomitantly reduced uPA production (10-20% parental) in response to calcitonin, forskolin and 8-bromo cAMP. The FIB1, FIB2 and FIB5 mutant cell lines had about 70% parental levels of cAMP-PK and the synthesis of uPA was 40-60% parental. Thus, cAMP-mediated induction of uPA showed a dependence on the absolute levels of cAMP-PK. However, uPA synthesis in response to phorbol-12-myristate-13-acetate by all of the mutants was similar to parental, which indicates that enzyme induction mediated by phorbol esters does not involve cAMP or cAMP-PK.     

Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V

Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. The extent to which the endocytic trafficking routes can contribute to such RTK hyperactivation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation were synergistic in stimulating phosphatidylinositol (PI) 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of PI 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in nontransformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor promoted macropinocytosis of both receptors and increased their activation in nontransformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.