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Cell Growth & Differentiation Vol. 13, 107-113, March 2002
© 2002 American Association for Cancer Research

Differential Regulation of a Novel Variant of the {alpha}6 Integrin, {alpha}6p1

Tracy L. Davis, Friederike Buerger and Anne E. Cress2

Department of Radiation Oncology, Arizona Cancer Center [T. L. D., F. B., A. E. C.], and the Department of Molecular and Cellular Biology [A. E. C.], University of Arizona, Tucson, Arizona 85724-5024


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We have reported previously the existence of an Mr 70,000 form of the {alpha}6 integrin called {alpha}6p in a variety of human epithelial cell lines. Four different experimental conditions were used to examine the regulation of {alpha}6 and {alpha}6p integrin. The production of the {alpha}6 integrin was decreased by 45% using a protein translation inhibitor (2.25 µM puromycin), whereas production of the {alpha}6p variant was unaffected. The {alpha}6p variant was decreased 60% by actin depolymerization (10 µM cytochalasin D) corresponding to a decrease in its surface expression, whereas {alpha}6 integrin production was unaffected. The {alpha}6p variant was resistant to endoglycosidase H treatment, whereas the {alpha}6 integrin was both sensitive and resistant to endoglycosidase H treatment, indicating retention in the endoplasmic reticulum and processing through the Golgi apparatus. Additionally, digestion by endoglycosidase F demonstrated both {alpha}6p and {alpha}6 integrin contained NH2-linked glycosylations and both shifted Mr ~10,000 on enzymatic digestion. Finally, inhibition of serine/threonine phosphatases by either calyculin A (15 nM) or okadaic acid (62 µM) did not affect {alpha}6p, whereas the production of {alpha}6 integrin was decreased by 50%. These data suggest that the production of the {alpha}6p variant is distinct from {alpha}6 integrin and may involve a post-translational processing event at the cell surface.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Integrins are signaling receptors that link the intracellular cytoskeleton to the extracellular matrix and play important roles in adhesion, migration, proliferation, signaling, differentiation, and cell survival (1, 2, 3, 4, 5, 6, 7, 8) . The {alpha}6 integrin is a laminin receptor in epithelial cells (9, 10, 11, 12, 13, 14) . Previously, studies demonstrated a loss of the {alpha}6ß4 heterodimer during prostate tumor progression (15, 16, 17) and a persistent expression of the {alpha}6ß1 integrin (18) . Additionally, expression of {alpha}6ß1 integrin is maintained in micrometastases (15 , 16 , 19, 20, 21) .

Our previous studies identified a novel Mr 70,000 variant of the {alpha}6 integrin, called {alpha}6p, for the Latin word parvus, in prostate carcinoma cell lines (22) . The variant paired with both ß1 and ß4 integrin subunits and was present in a number of epithelial carcinoma cell lines, as well as in a normal immortalized human keratinocyte cell line. Two-dimensional gel analysis and Western blotting data indicated the cytoplasmic light chain of the variant was identical to that of the full-length {alpha}6 integrin and that the primary alteration was a shortened extracellular heavy chain. The shortened extracellular domain was missing the putative ligand-binding domain contained within the ß-propeller (23, 24, 25) .

Adhesion to extracellular matrix proteins has been shown to play a role in cytoskeletal organization (26) . The {alpha}6ß1 integrin localizes to the focal adhesion, functioning to link the extracellular matrix to the actin cytoskeleton via the ß1 cytoplasmic domain for both signal transduction and mechanical stability of the cell during migration (8 , 15 , 27, 28, 29, 30) . This interaction has been shown to be important for integrin signaling and recruitment of scaffolding molecules, such as paxillin and filamentous-actin (31 , 32) .

The production of a variant form of the integrin, missing the ligand-binding region of the molecule, may influence these events. It is of particular interest to understand the circumstances surrounding the production of {alpha}6p and whether it is subject to similar regulatory controls as the production of the {alpha}6 integrin. We have extended our studies to examine the effect of known experimental perturbations of integrin function on the production of {alpha}6 and {alpha}6p integrin. The following experiments demonstrated that the {alpha}6 and {alpha}6p integrins responded differently to the inhibition of translation, the alteration of actin filaments, endoglycosidase digestion, and the action of serine/threonine phosphatase inhibitors. These data indicate that the mechanism of {alpha}6 and {alpha}6p production differs significantly. Furthermore, these data are consistent with the hypothesis that the {alpha}6p integrin is produced by a processing event after the molecule reaches the cell surface.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Production of {alpha}6 Integrin, but not {alpha}6p, was Translation Dependent.
Recently, Alais et al. (33) demonstrated that the expression of ß1 integrins could be regulated through translation-dependent mechanisms. Our previous studies indicated that the {alpha}6p variant was generated independent of a transcription event, such as an alternative mRNA splicing (22) . We used the translation inhibitor, puromycin, to determine the importance of translation on the production of both {alpha}6 and {alpha}6p integrins. The human prostate carcinoma DU145H cells were exposed to 2.25 µM puromycin for 18 h or DMSO vehicle. The {alpha}6 and {alpha}6p integrin proteins were identified at Mr 160,000 and 70,000 respectively, from a whole cell lysate (Fig. 1A)Citation . Puromycin treatment resulted in a 45% reduction of the {alpha}6 integrin as compared with the vehicle control (Fig. 1B)Citation . No effect on {alpha}6p integrin protein levels was observed. Although the production of the {alpha}6 integrin was dependent on translation, the level of the {alpha}6p variant was not affected by the inhibition of translation.



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  		Fig. 1. Expression of the {alpha}6 integrin, but not {alpha}6p, was dependent on translation. Human prostate carcinoma DU145H cells were treated with either 2.25 µM puromycin or DMSO vehicle for 18 h. Whole cell lysate (10–15 µg) was loaded and electrophoresed on a 7.5% polyacrylamide gel under nonreducing conditions. Proteins were transferred to PVDF membrane followed by Western blot analysis with anti-{alpha}6 integrin antibody, AA6A (A). Protein bands in A were quantified and graphed in Excel (B). Data shown were representative of three independent experiments.

 
Production of {alpha}6p, but not {alpha}6 Was Dependent on the Actin Cytoskeleton.
The actin cytoskeleton influences integrin behavior on the cell surface, such as integrin clustering, dispersal from focal adhesions, and integrin-mediated adhesion to extracellular matrix proteins (34) . Disruption of the actin cytoskeleton, but not the tubulin network, has been previously shown to inhibit {alpha}6ß1-mediated cell adhesion to laminin (35) . If the {alpha}6p variant was produced on the cell surface, one would expect the production of the variant to be dependent on the actin cytoskeleton. The human prostate carcinoma DU145H cell line was used for these studies because of the abundance of the {alpha}6ß1 and {alpha}6pß1 integrins (22) .

The actin staining in the DU145H cells revealed primarily a cortical staining pattern surrounding the periphery of the cells with few stress fibers, and treatment with cytochalasin D resulted in a loss of cortical actin replaced with perinuclear distribution of disorganized actin (data not shown). Microtubule networks were observed to radiate throughout the cytoplasm, originating from the microtubule organization centers near the nuclei of the DU145H cells, and treatment with nocodazole resulted in a loss of the tubulin network (data not shown). The total production of {alpha}6 and {alpha}6p integrins was examined after the addition of cytochalasin D. A time-dependent decrease in total {alpha}6p protein levels to ~60% of the control level at 18 h was observed, whereas the total {alpha}6 integrin protein levels were relatively unaltered (Fig. 2, A and B)Citation . The differential change in the {alpha}6 and {alpha}6p integrin proteins was apparent by 12 h postaddition of cytochalasin D, and by 18 h, the {alpha}6p integrin form had decreased to 60% of the vehicle control (DMSO; Fig. 2, A and BCitation ). The microtubule network was disrupted using 8 µM nocodazole, and the total amount of {alpha}6 and {alpha}6p integrins was examined to determine whether nonspecific effects on the cytoskeleton were responsible for the altered production of {alpha}6p variant (Fig. 2, C and D)Citation . No significant difference was observed in the total amount of the {alpha}6 and {alpha}6p integrin forms on depolymerization of the microtubules, suggesting that the tubulin network was not important for production of either {alpha}6 or {alpha}6p integrins.



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  		Fig. 2. Disruption of the actin cytoskeleton reduced total protein expression of {alpha}6p but not {alpha}6 integrin. Human prostate carcinoma DU145H cells were treated with either 10 µM cytochalasin D (A and B) or 8 µM nocodazole (C and D) over a 24-h period of time. Identical amounts of whole cell lysates (10 µg) were loaded and electrophoresed on a 7.5% polyacrylamide gel under nonreducing conditions. Proteins were transferred to a PVDF membrane followed by Western analysis for {alpha}6 integrin (A and C). The {alpha}6 and {alpha}6p protein bands were scanned and quantitated using Scion Image Analysis software and graphed in Excel (B and D). Data shown were representative of three experiments.

 
Cytochalasin D Reduced Cell Surface Expression of {alpha}6, {alpha}6p, and ß1 Integrins.
Because the data suggested that the {alpha}6p was produced on the cell surface, we next determined if the loss of the {alpha}6p production by cytochalasin D could be accounted for by the loss of {alpha}6p cell surface expression. To distinguish between surface and cytoplasmic integrin subunits, cell surface proteins were labeled using biotin before adding either cytochalasin D (10 µM) or nocodazole (8 µM) to the cells. Depolymerization of actin by cytochalasin D resulted in a significant loss of {alpha}6 and ß1 integrins from the cell surface to 36 and 30% of vehicle controls, respectively (Fig. 3, A and B)Citation , whereas the total production of {alpha}6 integrin was not affected (Fig. 2)Citation . In contrast, the surface protein levels of the {alpha}6p decreased to 67% of the control value (Fig. 3, A and B)Citation , and the total production of the {alpha}6p integrin was reduced to ~65% of the control value (Fig. 2)Citation . No change in cell surface {alpha}6, ß1, or {alpha}6p integrins was observed in cells treated with nocodazole (Fig. 3, A and B)Citation . These data indicated that cytochalasin D decreased the cell surface expression of {alpha}6, whereas the total level of {alpha}6 integrin was unaffected (Figs. 2Citation and 3Citation ). In contrast, both the {alpha}6p cell surface expression and the total {alpha}6p production was significantly decreased (Fig. 3)Citation . These data taken together suggested again that the {alpha}6p variant was produced at the cell surface, dependent on the actin cytoskeleton.



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  		Fig. 3. Disruption of the actin cytoskeleton significantly reduced cell surface expression of {alpha}6, {alpha}6p, and ß1 integrins. Surface changes of {alpha}6, ß1, and {alpha}6p were determined by surface of DU145H cells with biotin before treatment with either 10 µM cytochalasin D or 8 µM nocodazole for 18 h. Labeled cells were lysed, and 200 µg of total protein were used for immunoprecipitations with anti-{alpha}6 integrin antibody, J1B5. Samples were separated on a 7.5% polyacrylamide gel under nonreducing conditions. Proteins were transferred to PVDF membrane followed by incubation with HRP conjugated to streptavidin (A). Resulting {alpha}6, ß1, and {alpha}6p integrin protein bands were quantified and graphed (B).

 
Differential Intracellular Processing of the {alpha}6 and {alpha}6p Integrin.
It is known that the integrins can be modified after translation by glycosylation (2) . There are nine potential NH2-linked glycosylation sites contained in the {alpha}6 integrin (36 , 37) ; five are contained within exons 13–25, the region present within the {alpha}6p integrin (22) . The enzyme endoH3 is frequently used in combination with endoF to distinguish between complex and high-mannose oligosaccharides. Proteins sensitive to cleavage by endoH are not fully processed, i.e., retained in the Golgi apparatus, whereas proteins sensitive to endoF cleavage are fully processed by the Golgi (38) . We determined whether or not the {alpha}6p integrin variant was differentially glycosylated compared with the full-length {alpha}6 integrin. Human prostate carcinoma DU145H cells were lysed, immunoprecipitated with anti-{alpha}6 integrin antibody J1B5, and subjected to digestion with either endoH or endoF as detailed in "Materials and Methods." EndoH digestion resulted in the appearance of at least three {alpha}6 integrin intermediates, indicating the retention of these forms within the ER (Fig. 4)Citation . The majority of the {alpha}6 integrin was both endoH and endoF resistant, indicating successful passage through the ER and entrance into the medial Golgi compartment. In contrast, the {alpha}6p variant was not sensitive to endoH digestion but was sensitive to endoF (Fig. 4)Citation . No ER-retained forms of the {alpha}6p integrin were detected, although the {alpha}6p does contain high mannose type oligosaccharides. The data suggested that the variant may be produced after the molecule arrives at the cell surface, because the {alpha}6p was not processed through the ER and was not dependent on active protein translation.



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  		Fig. 4. The {alpha}6 integrin sensitivity to endoglycosidase treatment. Human prostate carcinoma DU145H cells were immunoprecipitated for {alpha}6 integrin using J1B5 antibody and then subjected to digestion with either endoH or endoF overnight at 37°C. Resulting protein samples were analyzed by 7.5% SDS-PAGE under nonreducing conditions followed by Western blot analysis using anti-{alpha}6 integrin antibody, AA6A. The migration of the molecular weight standards and integrins are indicated.

 
The {alpha}6, but not {alpha}6p Integrin, Was Altered by Serine/Threonine Phosphatase Inhibitors.
Inhibition of serine/threonine phosphatases using pharmacological inhibitors has been shown previously to regulate integrin phosphorylation (39 , 40) and integrin function (41, 42, 43) . Calyculin A is a potent inhibitor of protein phosphatase type 1 and 2A, whereas okadaic acid inhibits both, but it preferentially inhibits type 2A (44 , 45) .

To examine the role for protein phosphatase inhibitors on {alpha}6 and {alpha}6p integrins, calyculin A and okadaic acid were tested. Using 15 nM calyculin A to inhibit serine/threonine phosphatases, the total amount of {alpha}6 and {alpha}6p integrins was examined. After treatment for 6 h with 15 nM calyculin A, we observed a 50% decrease in total protein production of {alpha}6 integrin but only a 10% decrease in the variant {alpha}6p form (Fig. 5, A and B)Citation . Cells also were treated with 62 µM okadaic acid for 18 h. Two {alpha}6 integrin forms were observed after treatment (Fig. 5C)Citation . The molecular weight shift observed in the lower form was consistent with a dephosphorylated {alpha}6 integrin protein similar to that observed for {alpha}4 integrin (39) . There was a 2-fold increase of the faster migrating form of {alpha}6 integrin, with a corresponding 50% decrease in the slower migrating form (Fig. 5D)Citation . No alteration in electrophoretic mobility of {alpha}6p integrin was observed under the same experimental conditions. The pharmacological inhibitors used in this study were not toxic to the cells (data not shown).



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  		Fig. 5. Calyculin A and okadaic acid treatment of DU145H cells decreased {alpha}6 integrin protein levels but not {alpha}6p. Human prostate carcinoma DU145H cells were treated with 15 nM calyculin A over a 24-h period of time. Identical amounts of whole cell lysate (10) were loaded and electrophoresed on a 7.5% polyacrylamide gel under nonreducing conditions. Proteins were transferred to PVDF membrane followed by Western analysis for {alpha}6 integrin (A). Protein bands in A were scanned and quantified using Scion Image Analysis software and graphed in Excel (B). Data shown were representative of three independent experiments. DU145H cells were treated with 50 µM okadaic acid, the inactive analogue 1nor-okadaone, or vehicle (DMSO) for 18 h. Whole cell lysates were examined for {alpha}6 integrin protein expression as above (C). Resulting {alpha}6 and {alpha}6p bands from three independent experiments were quantified and graphed (D).

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Previous studies have indicated that the {alpha}6 integrin-containing heterodimer is altered in prostate carcinoma progression, shifting from the {alpha}6ß4 to {alpha}6ß1 integrin. Previously, we identified a novel variant of the {alpha}6 integrin, called {alpha}6p, which paired with both ß1 and ß4 subunits (22) . The variant was missing a large portion of the extracellular domain, including the postulated ligand-binding region, but retained an identical cytoplasmic light chain. Four different experimental strategies were used here to determine whether {alpha}6p and {alpha}6 were regulated in a similar or distinct manner. It was found that the response of the {alpha}6p and the {alpha}6 integrin to the experimental conditions was distinct, indicating a disassociation between the appearance of these two integrin forms.

The most striking difference in the forms was the susceptibility of the {alpha}6 integrin and the resistance of the {alpha}6p integrin to the inhibition of protein translation using puromycin. Our previous studies identified only one mRNA transcript for the {alpha}6 integrin in the DU145H cells (22) . One formal possibility to explain the production of a smaller version of {alpha}6 ({alpha}6p) was that an altered translation of the {alpha}6 mRNA occurred. Previous work has shown that isoforms of cell surface receptors can be generated by the selective use of internal ribosome entry sites or alternative translational start sites (46) . Recently, expression of ß1 integrin was altered by a translation-dependent mechanism (33) . Our studies indicated that production of the {alpha}6 integrin could be suppressed using an inhibitor of translation but that expression of the {alpha}6p variant was unaltered by translation inhibition. These data suggested that the {alpha}6p variant was generated through a post-translational mechanism. These data also may indicate that a larger "pool" of the wild-type {alpha}6 integrin exists relative to the {alpha}6p form. Inhibition of {alpha}6 production by puromycin may trigger processing of the {alpha}6 to the {alpha}6p form.

The integrin {alpha}6ß1 is processed after translation in the ER. The intracellular processing of the integrin can be monitored by determining the susceptibility of the protein to cleavage by endoH. Our results are similar to the findings of others that the {alpha}6 integrin contains both endoH-sensitive and -resistant forms, consistent with passage of the molecule through the ER and the Golgi compartment (47) . In contrast, the {alpha}6p integrin was resistant to endoH cleavage, indicating that it was not resident within the ER. Because the {alpha}6p is on the cell surface and does not traffic through the ER, it suggests that the protein was produced by a post-translational event occurring at the cell surface.

Integrins on the cell surface are known to be regulated by the cytoskeleton (26 , 32 , 48, 49, 50) , e.g., the actin cytoskeletal attachment to integrins is important for modulation of integrin clustering and dispersal from focal contacts and "inside-out" signaling. In this study, we observed that the cell surface abundance of the {alpha}6 and ß1 integrins was dependent on the actin cytoskeleton, whereas the total cellular production of the {alpha}6 integrin was unaltered. In contrast, both the production and the cell surface expression of the {alpha}6p form of the integrin were uniquely susceptible to actin depolymerization. These data combined with the resistance of the {alpha}6p to endoH suggests that the {alpha}6p variant formed after the integrin arrived on the surface of the cell.

The processing of cell surface receptors has been described previously as ectodomain shedding and plays an essential role in mammalian development (51) . We note with interest that collagen XVII/BP180, an epidermal adhesion molecule, exists as a full-length transmembrane protein and is processed into a Mr 120,000 ectodomain that is shed from the keratinocyte surface (52) . In addition, CD44, a specific adhesion receptor for hyaluronan, can be shed in a process that can be reduced by disruption of actin assembly with cytochalasin D (53) . Current work is under way to determine whether the {alpha}6p form of the integrin is generated in a manner similar to the process of ectodomain shedding. The data presented are consistent with a proteolytic processing of the {alpha}6 integrin on the cell surface to the {alpha}6p form. Experiments are under way to determine the nature of the protease activity involved. At the present, we know that broad-based metalloproteinase inhibitors are ineffective in blocking the {alpha}6p production (data not shown). This is in contrast to recent findings that the integrin {alpha}V can be processed by MT1-MMP (54) .

A final experimental approach to examine {alpha}6 and {alpha}6p function was the use of phosphatase inhibitors. Inhibition of serine/threonine phosphatases has been shown previously to decrease cell-cell adhesion (55 , 56) and integrin-dependent adhesion and motility (40, 41, 42, 43) . Inhibitors of serine/threonine phosphatases, such as okadaic acid and calyculin A, resulted in dephosphorylation of {alpha}4 integrin, resulting in high-avidity binding of VCAM-1 (39) . In our experiments, treatment with calyculin A and okadaic acid resulted in a differential alteration of the electrophoretic properties of the {alpha}6 integrin (Fig. 5)Citation . The {alpha}6 integrin in treated cells existed as a protein doublet. The {alpha}6p variant was not altered by treatment with serine/threonine phosphatase inhibitors. Although the significance of phosphorylation of the {alpha}6 integrin cytoplasmic domain is understood incompletely, it has been shown to induce tyrosine phosphorylation of paxillin and other unknown proteins on ligand binding (57 , 58) . Our results were suggestive that the cytoplasmic domain of the {alpha}6 integrin was responsive to a signaling event, whereas the {alpha}6p variant was not, despite having identical cytoplasmic domains (22) . In this instance, the {alpha}6p variant may play a dominant negative role. However, we note that ectopic expression of the {alpha}6 cytoplasmic domain alone in myoblasts is active in suppressing proliferation, induction of differentiation, and suppression of focal adhesion signaling (59 , 60) . These data would support the notion that an integrin lacking the extracellular ligand-binding domain may still retain a role in altering the cellular response to growth. Experiments are underway currently to determine the role of the {alpha}6p variant in the alteration of cellular adhesion and proliferation.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cell Culture.
Human prostate carcinoma cell line, DU145H, was isolated by us as described previously (19) . Cells were grown in IMDM (Life Technologies, Inc., Gaithersburg, MD) plus 10% fetal bovine serum and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Antibodies and Reagents.
Anti-{alpha}6 integrin antibodies were obtained as follows: GoH3, rat IgG2a (Accurate Chemicals, Westbury, NY; Ref. 61 ), J1B5, rat monoclonal was a generous gift from Dr. Caroline Damsky (University of California, San Francisco, CA; Ref. 62 ), and AA6A rabbit polyclonal, which was raised and purified using Bethyl Laboratories, Inc. (Montgomery, TX) specific for 16 amino acids (CIHAQPSDKERLTSDA) at the COOH terminus of the human {alpha}6A sequence (9) as done previously (11) . Cytoskeletal inhibitors cytochalasin D and nocodazole were obtained from Sigma Chemical Co. (St. Louis, MO). Serine/threonine phosphatase inhibitors were obtained as follows: calyculin A, Okadaic acid (Alexis Biochemicals, San Diego, CA), and inactive analogue 1-nor-okadaone (LC Laboratories, Woburn, MA). For inhibition of translation, puromycin was obtained (Sigma Chemical Co.).

Immunoprecipitations/Western Blot Analysis.
For immunoprecipitations, 200 µg of total protein lysate were used for each reaction and incubated with 35 µl of protein G Sepharose and 1 µg of antibody. The final volume of the lysate was adjusted to 500 µl with RIPA buffer [150 mM NaCl, 50 mM Tris, 5 mM EDTA, 1% (volume for volume) Triton X-100, 1% (w/v) deoxycholate, and 0.1% (w/v) SDS (pH 7.5)]. The mixture was rotated for 18 h at 4°C. After incubation, complexes were washed three times with cold RIPA and eluted in 2 x nonreducing sample buffer. Immunoprecipitation and whole cell lysate samples were boiled for 5 min before loading onto a 7.5% SDS-polyacrylamide gel for analysis. Proteins resolved in the gel were electrotransferred to Millipore Immobilon-P PVDF membrane (Millipore, Bedford, MA), incubated with either peroxidase-conjugated streptavidin or Western blotting antibodies plus secondary antibody conjugated to HRP and visualized by chemiluminescence (ECL Western Blotting Detection System; Amersham, Arlington Heights, IL), and exposed to film. Protein bands were quantitated using Scion Image Analysis software as described previously (63) and graphed using Excel software.

Alteration of {alpha}6 and {alpha}6p Integrins by Pharmacological Inhibitors.
Human prostate carcinoma DU145H cells were treated in serum-free IMDM media containing 0.1% BSA with drug (10 µM cytochalasin D, 8 µM nocodazole, 15 nM calyculin A, 62 µM okadaic acid, 62 µM 1-nor-okadaone, and 2.25 µM puromycin) for 18 h in the dark. For time courses, media were exchanged for serum-free IMDM containing 0.1% BSA at the start of the time course, and drug was added at appropriate time points. Cells were then collected by scraping, centrifuged for 5 min at 800 x g, and washed two times in HEPES buffer. Cell pellets were lysed in RIPA buffer with protease inhibitors and sonicated. Whole cell lysate (10–15 µg) was loaded and electrophoresed on a 7.5% SDS-polyacrylamide gel under nonreducing conditions. Proteins were transferred to PVDF membrane followed by Western analysis for {alpha}6 integrin with anti-{alpha}6 integrin antibody, AA6A. Protein bands for {alpha}6 and {alpha}6p were scanned and quantified using Scion Image Analysis software as described previously (63) and graphed using Excel software.

Surface changes of {alpha}6, ß1, and {alpha}6p were determined by surface biotinylation of DU145H cells followed by 18 h of drug treatment for cytochalasin D. For nocodazole studies, DU145H cells were labeled after the 18-h drug treatment. Biotinylated DU145H cells were lysed, and 200 µg of total protein were used for immunoprecipitations with anti-{alpha}6 integrin antibody, J1B5. Samples were analyzed as above, and PVDF membrane was incubated with HRP-streptavidin. Resulting protein bands for {alpha}6, ß1, and {alpha}6p from treated or vehicle samples were quantitated and graphed.

EndoH and EndoF Digestions.
For digestions, 200 µg of whole cell lysate were first immunoprecipitated overnight with anti-{alpha}6 integrin antibody, J1B5, in microcentrifuge tubes. The following day, the beads were washed three times with RIPA buffer, and the sample was resuspended in 35 µl of 2 x nonreducing sample buffer containing 4 mM CaCl2 plus 1 mUnit either endoH or endoF (obtained from Sigma Chemical Co.), which had been diluted in 10% glycerol. Tubes containing reactions were placed in a shaking hot water bath at 37°C overnight. The following morning, the samples were analyzed by 7.5% SDS-PAGE under nonreducing conditions followed by Western blot analysis using anti-{alpha}6 integrin antibody, AA6A.


   Acknowledgments
 
We thank Dr. Caroline Damsky for the contribution of the J1B5 hybridoma and discussions with Drs. S. Dedhar and J. C. R. Jones.


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

1 Supported by Grants CA56666 and CA75152 from the National Cancer Institute. Back

2 To whom requests for reprints should be addressed, at The Arizona Cancer Center, The University of Arizona, 1501 N. Campbell Avenue, Tucson, AZ 85724-5024. Phone: (520) 626-7553; Fax: (520) 626-4979; E-mail: acress{at}azcc.arizona.edu Back

3 The abbreviations used are: endoH, endoglygosidase H; endoF, endoglycosidase F; ER, endoplasmic reticulum; IMDM, Iscove’s modified Dulbecco’s medium; RIPA, radioimmunoprecipitation assay; PVDF, polyvinylidene fluoride; HRP, horseradish peroxidase. Back

Received for publication 7/23/01. Revision received 12/20/01. Accepted for publication 1/ 4/02.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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