Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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--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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-(2633)]
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).
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).
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
|
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.
|
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).
|
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.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Backer, J. M.,
C. R. Kahn,
D. A. Cahill,
A. Ullrich,
and
M. F. White.
Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor beta-subunit.
J. Biol. Chem.
265:
16450-16454,
1990
2.
Barak, L. S.,
M. Tiberi,
N. J. Freedman,
M. M. Kwatra,
R. J. Lefkowitz,
and
M. G. Caron.
A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated 2-adrenergic receptor sequestration.
J. Biol. Chem.
269:
2790-2795,
1994
3.
Benya, R. V.,
Z. Fathi,
J. F. Battey,
and
R. T. Jensen.
Serines and threonines in the gastrin-releasing peptide receptor carboxyl terminus mediate internalization.
J. Biol. Chem.
268:
20285-20290,
1993
4.
Benya, R. V.,
T. Kusui,
J. F. Battey,
and
R. T. Jensen.
Chronic desensitization and down-regulation of the gastrin-releasing peptide receptor are mediated by a protein kinase C-dependent mechanism.
J. Biol. Chem.
270:
3346-3352,
1995
5.
Bredt, D. S.,
R. J. Mourey,
and
S. H. Snyder.
A simple, sensitive, and specific radioreceptor assay for inositol 1,4,5-trisphosphate in biological tissues.
Biochem. Biophys. Res. Commun.
159:
976-982,
1989[Medline].
6.
Chen, W. J.,
J. L. Goldstein,
and
M. S. Brown.
NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor.
J. Biol. Chem.
265:
3116-3123,
1990
7.
De Toledo, C. F.,
B. F. Roettger,
C. Morys-Wortmann,
W. E. Schmidt,
and
L. J. Miller.
Cellular handling of unoccupied and agonist-stimulated cholecystokinin receptor determined by immunolocalization.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G488-G497,
1997
8.
Eason, M. G.,
M. T. Jacinto,
C. T. Theiss,
and
S. B. Liggett.
The palmitoylated cysteine of the cytoplasmic tail of 2A-adrenergic receptors confers subtype-specific agonist-promoted downregulation.
Proc. Natl. Acad. Sci. USA
91:
11178-11182,
1994
9.
Ferguson, S. S. G.,
W. E. Downey III,
A. M. Colapietro,
L. S. Barak,
L. Ménard,
and
M. G. Caron.
Role of -arrestin in mediating agonist-promoted G protein-coupled receptor internalization.
Science
271:
363-366,
1996[Abstract].
10.
Gates, L. K.,
C. D. Ulrich,
and
L. J. Miller.
Multiple kinases phosphorylate the pancreatic cholecystokinin receptor in an agonist-dependent manner.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G840-G847,
1993
11.
Go, W. Y.,
B. F. Roettger,
E. L. Holicky,
E. M. Hadac,
and
L. J. Miller.
Quantitative dynamic multicompartmental analysis of cholecystokinin receptor movement in a living cell using dual fluorophores and reconstruction of confocal images.
Anal. Biochem.
247:
210-215,
1997[Medline].
12.
Goodman, O. B., Jr.,
J. G. Krupnick,
F. Santini,
V. V. Gurevich,
R. B. Penn,
A. W. Gagnon,
J. H. Keen,
and
J. L. Benovic.
-Arrestin acts as a clathrin adaptor in endocytosis of the
2-adrenergic receptor.
Nature
383:
447-450,
1996[Medline].
13.
Hadac, E. M.,
D. V. Ghanekar,
E. L. Holicky,
D. I. Pinon,
R. W. Dougherty,
and
L. J. Miller.
Relationship between native and recombinant cholecystokinin receptors: role of differential glycosylation.
Pancreas
13:
130-139,
1996[Medline].
14.
Holtmann, M. H.,
B. F. Roettger,
D. I. Pinon,
and
L. J. Miller.
Role of receptor phosphorylation in desensitization and internalization of the secretin receptor.
J. Biol. Chem.
271:
23566-23571,
1996
15.
Hunyady, L.,
M. Bor,
T. Balla,
and
K. J. Catt.
Identification of a cytoplasmic Ser-Thr-Leu motif that determines agonist-induced internalization of the AT1 angiotensin receptor.
J. Biol. Chem.
269:
31378-31382,
1994
16.
Hunyady, L.,
M. Bor,
A. J. Baukal,
T. Balla,
and
K. J. Catt.
A conserved NPLFY sequence contributes to agonist binding and signal transduction but is not an internalization signal for the type 1 angiotensin II receptor.
J. Biol. Chem.
270:
16602-16609,
1995
17.
Kawate, N.,
and
K. M. J. Menon.
Palmitoylation of luteinizing hormone/human choriogonadotropin receptors in transfected cells. Abolition of palmitoylation by mutation of Cys-621 and Cys-622 residues in the cytoplasmic tail increases ligand-induced internalization of the receptor.
J. Biol. Chem.
269:
30651-30658,
1994
18.
Kennedy, M. E.,
and
L. E. Limbird.
Mutations of the 2A-adrenergic receptor that eliminate detectable palmitoylation do not perturb receptor-G-protein coupling.
J. Biol. Chem.
268:
8003-8011,
1993
19.
Klueppelberg, U. G.,
L. K. Gates,
F. S. Gorelick,
and
L. J. Miller.
Agonist-regulated phosphorylation of the pancreatic cholecystokinin receptor.
J. Biol. Chem.
266:
2403-2408,
1991
20.
Lutz, M. P.,
D. I. Pinon,
L. K. Gates,
S. Shenolikar,
and
L. J. Miller.
Control of cholecystokinin receptor dephosphorylation in pancreatic acinar cells.
J. Biol. Chem.
268:
12136-12142,
1993
21.
Lutz, M. P.,
S. L. Sutor,
R. T. Abraham,
and
L. J. Miller.
A role for cholecystokinin-stimulated protein tyrosine phosphorylation in regulated secretion by the pancreatic acinar cell.
J. Biol. Chem.
268:
11119-11124,
1993
22.
Moench, S. J.,
J. Moreland,
D. H. Stewart,
and
T. G. Dewey.
Fluorescence studies of the location and membrane accessibility of the palmitoylation sites of rhodopsin.
Biochemistry
33:
5791-5796,
1994[Medline].
23.
Moffett, S.,
B. Mouillac,
H. Bonin,
and
M. Bouvier.
Altered phosphorylation and desensitization patterns of a human 2-adrenergic receptor lacking the palmitoylated Cys341.
EMBO J.
12:
349-356,
1993[Abstract].
24.
Munson, P. J.,
and
D. Rodbard.
LIGAND: a versatile computerized approach for characterization of ligand-binding systems.
Anal. Biochem.
107:
220-239,
1980[Medline].
25.
Mutt, V.
Cholecystokinin: isolation, structure, and functions.
In: Gastrointestinal Hormones, edited by G. B. J. Glass. New York: Raven, 1980, p. 169-221.
26.
Namba, T.,
Y. Sugimoto,
M. Negishi,
A. Irie,
F. Ushikubi,
A. Kakizuka,
S. Ito,
A. Ichikawa,
and
S. Narumiya.
Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity.
Nature
365:
166-170,
1993[Medline].
27.
Ng, G. Y.,
B. Mouillac,
S. R. George,
M. Caron,
M. Dennis,
M. Bouvier,
and
B. F. O'Dowd.
Desensitization, phosphorylation and palmitoylation of the human dopamine D1 receptor.
Eur. J. Pharmacol. Mol. Pharmacol. Sect.
267:
7-19,
1994[Medline].
28.
Ozcelebi, F.,
and
L. J. Miller.
Phosphopeptide mapping of cholecystokinin receptors on agonist-stimulated native pancreatic acinar cells.
J. Biol. Chem.
270:
3435-3441,
1995
29.
Ozcelebi, F.,
R. V. Rao,
E. Holicky,
B. J. Madden,
D. J. McCormick,
and
L. J. Miller.
Phosphorylation of cholecystokinin receptors expressed on Chinese hamster ovary cells: similarities and differences relative to native pancreatic acinar cells.
J. Biol. Chem.
271:
3750-3755,
1996
30.
Pohl, M.,
S. Silvente-Poirot,
J. R. Pisegna,
N. I. Tarasova,
and
S. A. Wank.
Ligand-induced internalization of cholecystokinin receptors. Demonstration of the importance of the carboxyl terminus for ligand-induced internalization of the rat cholecystokinin type B receptor but not the type A receptor.
J. Biol. Chem.
272:
18179-18184,
1997
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-2633].
J. Biol. Chem.
263:
5295-5300,
1988
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
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
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
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