Identification of a domain in the carboxy terminus of CCK receptor that affects its intracellular trafficking

William Y. Go, Eileen L. Holicky, Elizabeth M. Hadac, Rammohan V. Rao, and Laurence J. Miller

Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The carboxy-terminal region of many guanine nucleotide-binding protein (G protein)-coupled receptors contains important regulatory sequences such as an NP(x)2-3Y motif, a site of fatty acid acylation, and serine- and threonine-rich domains. The type A CCK receptor contains all of these, yet their significance has not been examined. We have, therefore, constructed a series of receptor site mutants and truncations that interfere with each of these motifs and expressed each in Chinese hamster ovary cells where they were studied for radioligand binding, cell signaling, receptor internalization, and intracellular trafficking. Each construct was synthesized and transported appropriately to the cell surface, where CCK bound with high affinity, elicited an inositol 1,4,5-trisphosphate response, and resulted in internalization and normal trafficking. Thus modification or elimination of each of these established sequence motifs had no substantial effect on any of these parameters of receptor and cellular function. However, an additional construct that truncated the carboxy terminus, eliminating an additional 15-amino-acid segment devoid of any currently recognized sequence motifs, resulted in a marked change in receptor trafficking, with all other parameters of receptor function normal. This mutant receptor construct was delayed at the stage of early endosomes, delaying its progress to the lysosome-enriched perinuclear compartment from the rapid time course followed by wild-type receptor and all of the other constructs. It is proposed that this region of the CCK receptor tail contains a new motif important for intracellular receptor trafficking.

G protein-coupled receptor; receptor internalization; receptor trafficking; endocytosis; cholecystokinin

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE CARBOXY-TERMINAL DOMAIN of several guanine nucleotide-binding protein (G protein)-coupled receptors has been reported to influence the function and regulation of those receptors (2, 15, 26). Key contributions to signaling and receptor internalization have been localized to the NP(x)2-3Y motif in the juxtamembranous location (2, 16), sites of fatty acid acylation (palmitoylation) (8, 17, 23, 27), and serine- and threonine-rich domains (3, 15). Effects of these domains and motifs, however, have been inconsistent, depending on the specific receptor and cell system studied (14, 18, 33, 37). Receptor domains have not previously been recognized to affect the trafficking of endosomes carrying them postinternalization.

The type A CCK receptor is a member of the rhodopsin-beta -adrenergic receptor family of G protein-coupled receptors, which is expressed on the pancreatic acinar cell, smooth muscle cells of the gallbladder and sites within the gastrointestinal tract, and select neurons in the peripheral and central nervous system (25). It has great physiological and potential pathophysiological relevance. It is an interesting receptor to study because it possesses all of these described carboxy-terminal motifs, yet the function of each has not been previously explored in detail. Furthermore, our previous work has established a series of useful tools and background information for the structure, function, and regulation of this receptor (19, 29, 31, 34, 35, 39).

In this study, we have investigated the functional roles of each of these carboxy-terminal domains and motifs within the CCK receptor. For this, we constructed a series of site-mutants and truncated receptor constructs and established Chinese hamster ovary (CHO) cell lines stably expressing each. Radioligand binding, signaling, and receptor internalization and intracellular trafficking were studied in each cell line. Of particular interest, none of these recognized regulatory regions had any impact in any of these receptor functions. The only domain within the carboxy-terminal tail of the CCK receptor was a 15-residue-long segment which did not possess any of these recognized motifs and which was positioned between the site of palmitoylation and the serine- and threonine-rich domain at the end of the receptor tail. When this domain was eliminated by truncation, ligand binding, signaling, and initial movement off the cell surface continued to be normal; however, intracellular trafficking was clearly delayed. We postulate that this domain contributes important determinants for molecular interactions key for movement of receptor-bearing vesicles deep within the cell. This provides an additional previously unrecognized regulatory influence of the receptor tail and suggests that the cargo of endocytic vesicles can influence their movement.

    METHODS
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Methods
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Materials. Synthetic sulfated CCK-octapeptide (CCK-8) was purchased from Peninsula Laboratories (Belmont, CA). Radioiodinated and fluorescent analogs of CCK, which we have previously established and characterized (32, 35), were freshly prepared in our laboratory. The radioligand I-D-Tyr-Gly-[(Nle28,31)CCK-(26---33)] was purified by reverse-phase HPLC to a specific radioactivity of 2,000 Ci/mmol and has been shown to represent a full agonist and to bind to receptor with identical affinity to native hormone (32). The fluorescent ligand, rhodamine-Gly-[(Nle28,31)CCK-(26---33)] (Rho-CCK), was also purified to homogeneity, and represents a full agonist that binds normally (19). Dioctadecyloxacarbocyanine perchlorate (DiO) was purchased from Molecular Probes (Eugene, OR).

The CCK receptor-bearing cell line (CHO-CCKR), which was previously established and fully characterized (13), was used as a wild-type receptor-bearing control. It was cultured on tissue culture plastic in Ham's F-12 medium in a humidified incubator at 37°C in an atmosphere of 5% CO2. Cells were passaged twice weekly. For experiments, cells were lifted mechanically, triturated, and washed with appropriate medium before use.

CCK receptor mutagenesis. For mutagenesis the wild-type rat type A CCK receptor construct in m13mp18 (Pharmacia Biotech, Piscataway, NJ) was used as a template (13). Mutants were constructed using the Sculptor in vitro mutagenesis system (Amersham Life Science, Arlington Heights, IL). The constructs were then subcloned into the pcDNA3 eukaryotic expression vector (InVitrogen, San Diego, CA).

Constructs are described in Fig. 1. Truncated constructs were prepared by incorporating a termination codon in the relevant position. Site mutants were prepared by changing the relevant codon appropriately. The identities of all constructs were proven by direct dideoxynucleotide DNA sequence analysis (36).


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Fig. 1.   Schematic representation of sequences of wild-type rat CCK receptor and mutant and truncated constructs used in this work.

Establishment of CCK receptor mutant-bearing cell lines. CHO cells (American Type Culture Collection, Rockville, MD), which do not express any detectable wild-type CCK type A or B receptors (13, 35), were used for transfection. This was accomplished by lipofection with Lipofectin (Life Technologies, Gaithersburg, MD), using 15 µg of each construct per 100-mm dish. After 48-72 h neomycin-resistant cells were selected by adding 1 mg/ml G418. Surviving cells expressing an appropriate receptor density were then selected by sequential rounds of fluorescence-activated cell sorting after incubation with a fluorescein-conjugated CCK receptor ligand, as previously described (13). Cells were cultured similarly to the CHO-CCKR cell line described previously.

CCK receptor binding. CCK radioligand binding was performed using conditions previously established (13). Each tube contained 3-5 pM radioligand and ~1.0-1.5 million cells in 0.5 ml Krebs-Ringer-HEPES medium containing (in mM) 25 HEPES, pH 7.4, 1 KH2PO4, 104 NaCl, 5 KCl, 2 CaCl2, and 1.2 MgSO4, and 0.2% BSA, and 0.01% soybean trypsin inhibitor, with each condition set up in duplicate. Binding was allowed to proceed for 60 min at room temperature, followed by separation of bound from free radioligand using a Skatron cell harvester with receptor-binding filtermats. Receptor binding affinity and density were determined by analysis using the LIGAND program of Munson and Rodbard (24). Data were graphed using Prism software (GraphPad Software, San Diego, CA).

Cell signaling. Agonist-stimulated cell signaling was assessed for each CCK receptor construct as we previously described for the wild-type receptor (33). Cells were studied in the basal state and after stimulation with CCK for 5 s, with each condition studied in duplicate. Cellular content of inositol 1,4,5-trisphosphate (IP3) was determined using a radioreceptor binding assay that is specific for this biologically active isomer of IP3 and is sensitive to 0.1 pmol of IP3 per tube (5).

CCK receptor internalization. CCK-stimulated CCK receptor internalization was studied as we previously reported, following fluorescent ligand movement in the cell using morphological techniques (35). This has been carefully validated, using receptor antibody, to be certain that this reflects movement of both ligand and receptor (7). Furthermore, a special procedure involving the use of dual fluorescent probes, one to label the receptor (a rhodamine-conjugated CCK analog) and one for registration of the location of the plasmalemma (DiO) was utilized (11). As previously validated (7), cells that had been incubated with fluorescent ligand at 37°C for given periods of time were fixed with 2% paraformaldehyde and analyzed by confocal laser scanning microscopy. The entire volume of the cell was scanned, and the images were used for three-dimensional reconstruction. Full quantitative reconstruction and analysis were performed on three cells for each time point in each experiment, with these cells visually typical of images representing a minimum of 30 cells treated similarly. Fluorescence above background at the level of the plasma membrane, in subplasma membrane endocytic vesicles, and in the lysosome-enriched perinuclear compartment were then quantified using the algorithm and formulas previously established.

Data analysis. All experimental conditions were studied in a minimum of three independent experiments. Data are expressed as means ± SE, with differences examined using Student's t-test for unpaired values. Significant differences were considered at the P < 0.05 level.

    RESULTS
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Abstract
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Methods
Results
Discussion
References

Each of the CCK receptor constructs depicted in Fig. 1 was appropriately produced by the respective CHO cell line, with biosynthesis and processing resulting in the delivery of a receptor to the cell surface, which bound radioligand and elicited normal signaling in response to CCK.

CCK competed for the CCK radioligand binding to each of the construct-bearing cell lines (Fig. 2). A summary of these data is shown in Table 1. No construct had a binding affinity for CCK that was statistically different from that for the wild-type receptor. Receptor density was somewhat reduced in the mutant receptor-bearing cells relative to the wild-type receptor-bearing cells.


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Fig. 2.   Binding characterization of each of the CCK receptor constructs. Shown are the abilities of CCK to compete for binding of the CCK-like radioligand to intact CHO cell lines expressing each of the mutant and truncated receptor constructs. Data are means ± SE for a minimum of 3 independent experiments. Curves best fitting the data have been determined by Prism software.

                              
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Table 1.  

CCK stimulated an IP3 response in each of the CCK receptor constructs as well (Fig. 3). The concentration of CCK that elicited EC50 for each is listed in Table 1. The differences between these responses did not reach statistical significance, and the absolute magnitude of the IP3 responses correlated with receptor densities determined in the radioligand binding studies.


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Fig. 3.   Signaling by each of the CCK receptor (CCKR) constructs. Shown are concentration-response curves for the abilities of CCK to stimulate inositol 1,4,5-trisphosphate (IP3) responses in CHO cell lines expressing each of the mutant and truncated receptor constructs. WT, wild type. Data are means ± SE for a minimum of 3 independent experiments. Curves have been determined and drawn according to Prism software.

Figure 4 illustrates the sensitivity of the fluorescent morphological technique for tracking ligand-occupied receptor through the most proximal cellular compartments after binding. This demonstrates the ability of the fluorescent morphological technique to distinguish plasmalemmal from subplasmalemmal localization of the ligand. This method has been fully validated and explained, with the full derivation of the method of quantitation reported elsewhere (11).


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Fig. 4.   Morphological assessment of internalization of ligand occupying the CCK receptor. Shown are representative images from the first 5 min of occupation of the CCK receptor on CHO-CCKR cells with rhodamine-conjugated CCK, as well as labeling of the plasma membrane with dioctadecyloxacarbocyanine perchlorate (DiO), and an integrated red-green-blue (RGB) image. Initially, the ligand-occupied receptor was within the plasma membrane, and it subsequently aggregated in clathrin-coated pits and moved into the classical endocytic compartments. Correlates to these cellular positions are shown in low-power electron micrographs of colloidal gold-conjugated CCK, using methodology previously established in the laboratory and reported (35).

Figure 5 shows representative images from the internalization studies for each of the CCK receptor constructs. Quantitation of data from this series of experiments to explore the movement of the agonist-bound receptor over time through the established cellular compartments is shown in Fig. 6. Indeed, whereas all of the receptor constructs were internalized after agonist occupation and warming to 37°C, one construct (CCKR, 1-408) was significantly delayed in moving through the endosome-enriched subplasmalemmal vesicular compartment (P = 0.004 at 5 min) and arriving in the lysosome-enriched perinuclear compartment (P values of 0.02 or less beginning at 10 min). It is noteworthy that the initial movement of this construct off the cell surface was normal, demonstrating that the more widespread use of acid washing to evaluate internalization would have been entirely normal, missing this important phenomenon. All other receptor constructs were indistinguishable from wild-type receptor in all of their movements.


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Fig. 5.   Morphological assessment of agonist-occupied CCK receptor internalization. Shown are representative images from the internalization time courses for wild-type (WT) CCK receptor and for each of the CCK receptor mutants and truncated constructs. Top 3 rows show typical pseudocolor images representing integrated RGB images and those acquired for the plasma membrane marker DiO using excitation at 488 nm and detecting emission with a 530 ± 15 nm band-pass filter and for the rhodaminated CCK ligand, using excitation at 568 nm and detection of emission using a 590 nm long-pass filter. For each construct, we show only the rhodamine ligand images. All images are representative of a minimum of 3 independent series of studies.


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Fig. 6.   Quantitation of internalization and cellular trafficking of fluorescent agonist-occupied CCK receptor constructs. Shown are quantitation of movement of the fluorescent ligand from plasma membrane, to punctate vesicular compartments in subplasma membrane area, to lysosome-enriched perinuclear compartment. * Significant differences from the wild-type receptor (P < 0.05). Data represent means ± SE for a minimum of 3 series of experiments.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Molecular interactions represent the most basic and apparently ubiquitous mechanism for propagation of signaling cascades within cells and for the regulated movement of cellular organelles. Recognition of this has resulted in the elucidation of a number of sequence motifs that appear to be key in specific processes. The carboxy-terminal domain of the heptahelical G protein-coupled receptors is a region particularly rich in such motifs. Among these are the NP(x)2-3Y motif in the juxtamembranous domain, site(s) of fatty acid acylation (palmitoylation), and serine- and threonine-rich domains. In specific members of this superfamily, each of these motifs has been reported to affect either receptor binding, signaling, or internalization (2, 15, 23). Of particular interest, there are other examples in the same group of molecules in which the same motif appearing in the same general domain has had no effect on the same parameters (14, 18, 33, 37). Our understanding of these processes will clearly require the analysis of additional examples and better elucidation of molecular mechanisms.

The CCK receptor, expressing all of these recognized motifs and previously having its phosphorylation (10, 19, 20, 28, 29) and internalization (34, 35) extensively characterized, represents an ideal target to study. It is particularly interesting that none of the motifs listed above appears to have any substantial effect on CCK receptor binding, signaling, or internalization. This was demonstrated in receptor constructs in which the NP(x)2-3Y motif was disrupted by mutagenesis of the tyrosine to an alanine, the site of fatty acid acylation of vicinal cysteines was removed by mutation to alanine residues, and the serine- and threonine-rich distal domain of the carboxy terminus was removed by truncation.

In contrast to these established motifs, an unexpected domain distal to the site of fatty acid acylation and proximal to the serine- and threonine-rich terminal tail of the CCK receptor, which does not have any known recognition motif, did affect receptor trafficking within the cell. It is postulated that this 15-amino-acid segment contains a new and important motif that is involved in the bimolecular interactions that move early endosomes deeper into the endocytic cascade toward the lysosome. It is noteworthy how acidic this segment is, but it has no clear sequence homology to domains of other receptors in this superfamily or to other intracellular protein sequences in existing public databases.

Perhaps there are instructive insights to be gained even from negative data. The NPXY motif found in the juxtamembranous region of a number of single transmembrane receptors, such as receptors for low-density lipoprotein, insulin, and epidermal growth factor, and felt to be a key site of tyrosine phosphorylation, first focused attention on this sequence (1, 6, 38). We now know that phosphorylation of that tyrosine leads to a molecular association with the phosphotyrosine-binding domain of Shc (41) and may contribute to its ultimate entry into the endocytic cascade. Many G protein-coupled receptors have a similar motif, but one containing two or three "spacer" residues [NP(x)2-3Y]. Although there is no similar evidence that the tyrosine in those receptors is phosphorylated, mutagenesis of that residue in the beta 2-adrenergic receptor interferes with its internalization (2). This does not occur in response to an analogous mutation in the gastrin-releasing peptide receptor (37). We have also not been able to demonstrate this effect for the CCK receptor. Of note, we have also not observed any tyrosine phosphorylation within the CCK receptor, despite agonist occupation of this receptor leading to stimulated tyrosine phosphorylation of other molecules within the cell (21).

Fatty acid acylation of cysteine residues in the carboxy-terminal tail only exists on a subset of G protein-coupled receptors, with many not possessing such a residue. As a potential regulatory motif, this can therefore not have a necessary and required function. In rhodopsin, it has been shown that this posttranslational modification brings the affected cysteine into contact with the plasmalemma to effect a fourth intracellular loop (22). Although the acylation of this residue appears to be constitutive and unregulated in many receptors, there is some evidence that in select receptors, the acylation reaction and the cleavage of the fatty acid occur in response to agonist stimulation (27). That leads to the dissociation from the membrane of a domain, which when in a juxtamembranous position facilitates G protein coupling and initiation of signaling (40).

Although we have evidence that the CCK receptor is palmitoylated, there is no published evidence for agonist regulation of this posttranslational modification of this receptor. Perhaps the absence of effect of mutagenesis of the key conserved vicinal cysteine residues in the tail seen in this work is consistent with this mechanism not being operative in this receptor.

Receptor tail domains that are rich in serine and threonine residues presumably affect internalization by becoming phosphorylated (4, 15). This can be a site of interaction with arrestin-like molecules (9), which can in turn result in a binding association with clathrin (12), a prominent coat protein involved in receptor-mediated endocytosis. We know, however, that the tail domain of the CCK receptor, despite being quite rich in serine and threonine, is the site of <5% of agonist-stimulated phosphorylation of this receptor (28). Ninety-five percent of agonist-stimulated phosphorylation of the CCK receptor occurs on serine and threonine residues within the third intracellular loop (19, 28). It is also noteworthy that phosphorylation is not necessary for internalization or normal intracellular trafficking of this receptor (33). That insight came from a CCK receptor mutation of the two key sites of phosphorylation within the third loop that results in no agonist-stimulated phosphorylation, but normal ligand binding, signaling, and receptor internalization (33).

Thus it appears that G protein-coupled receptors have a large and diverse repertoire of regulatory motifs. Some may play a direct role in subsequent intermolecular events, whereas others likely work via allosteric effects. It is likely that all share a relationship to conformational change. The conformational change in agonist-occupied receptors leads to exposure of new receptor domains that may have previously been hidden, with their exposure and/or posttranslational modification propagating activity cascades.

It should be emphasized that the morphological approach used in this work is very powerful and may identify receptor domains that would not have been found using more traditional techniques. Perhaps an example of this is the recent report (30) in which a truncated form of the CCK type A receptor with the carboxy-teminal 45 residues eliminated was found to have no effect on receptor internalization, determined by potassium thiocyanate stripping of surface radioligand. Although that observation was confirmed by morphological analysis of a green fluorescent protein conjugate of the receptor, the details of trafficking in that work were not focused on or quantified. The apparent difference from that work could also reflect the cellular environment in which these receptors were expressed, with NIH/3T3 cells used there and CHO cells used in the present study.

From the current work, we know that several established motifs are not critical for CCK receptor function and regulation. We also know that a previously unrecognized region must contain a motif that directly or indirectly affects receptor trafficking of this receptor in the CHO cell. This helps to expand our understanding of this important receptor and will likely have much more widespread significance.

    ACKNOWLEDGEMENTS

We acknowledge the help and advice of J. Tarara, D. Pinon, S. M. Kuntz, and Dr. B. Roettger and the excellent secretarial assistance of S. Erickson.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and the Kidney Diseases Grant DK-32878, the Fiterman Foundation, and the American Gastroenterological Association Summer Student Award.

Address for reprint requests: L. J. Miller, Center for Basic Research in Digestive Diseases, Guggenheim 17, Mayo Clinic, Rochester, MN 55905.

Received 4 November 1997; accepted in final form 11 March 1998.

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Abstract
Introduction
Methods
Results
Discussion
References

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31.   Powers, S. P., D. Fourmy, H. Gaisano, and L. J. Miller. Intrinsic photoaffinity labeling probes for cholecystokinin (CCK)-gastrin family receptors D-Tyr-Gly-[Nle28,31,pNO2-Phe33 CCK-26---33]. J. Biol. Chem. 263: 5295-5300, 1988[Abstract/Free Full Text].

32.   Powers, S. P., D. I. Pinon, and L. J. Miller. Use of N,O-bis-FMOC-D-Tyr-ONSu for introduction of an oxidative iodination site into cholecystokinin family peptides. Int. J. Pept. Protein Res. 31: 429-434, 1988[Medline].

33.   Rao, R. V., B. F. Roettger, E. M. Hadac, and L. J. Miller. Roles of cholecystokinin receptor phosphorylation in agonist-stimulated desensitization of pancreatic acinar cells and receptor-bearing Chinese hamster ovary cholecystokinin receptor cells. Mol. Pharmacol. 51: 185-192, 1997[Abstract/Free Full Text].

34.   Roettger, B. F., R. U. Rentsch, E. M. Hadac, E. H. Hellen, T. P. Burghardt, and L. J. Miller. Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell. J. Cell Biol. 130: 579-590, 1995[Abstract].

35.   Roettger, B. F., R. U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J. M. Larkin, and L. J. Miller. Dual pathways of internalization of the cholecystokinin receptor. J. Cell Biol. 128: 1029-1042, 1995[Abstract].

36.   Sanger, F., S. Nicklen, and A. R. Coulson. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977[Abstract].

37.   Slice, L. W., H. C. Wong, C. Sternini, E. F. Grady, N. W. Bunnett, and J. H. Walsh. The conserved NPXnY motif present in the gastrin-releasing peptide receptor is not a general sequestration sequence. J. Biol. Chem. 269: 21755-21761, 1994[Abstract/Free Full Text].

38.   Thies, R. S., N. J. Webster, and D. A. McClain. A domain of the insulin receptor required for endocytosis in rat fibroblasts. J. Biol. Chem. 265: 10132-10137, 1990[Abstract/Free Full Text].

39.   Ulrich, C. D., I. Ferber, E. Holicky, E. Hadac, G. Buell, and L. J. Miller. Molecular cloning and functional expression of the human gallbladder cholecystokinin A receptor. Biochem. Biophys. Res. Commun. 193: 204-211, 1993[Medline].

40.   Wade, S. M., H. M. Dalman, S.-Z. Yang, and R. R. Neubig. Multisite interactions of receptors and G proteins: enhanced potency of dimeric receptor peptides in modifying G protein function. Mol. Pharmacol. 45: 1191-1197, 1994[Abstract].

41.   Zhou, S., B. Margolis, M. Chaudhuri, S. E. Shoelson, and L. C. Cantley. The phosphotyrosine interaction domain of SHC recognizes tyrosine-phosphorylated NPXY motif. J. Biol. Chem. 270: 14863-14866, 1995[Abstract/Free Full Text].


Am J Physiol Gastroint Liver Physiol 275(1):G56-G62
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