©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Phenylalanine 346 in the Rat Growth Hormone Receptor as Being Critical for Ligand-mediated Internalization and Down-regulation (*)

(Received for publication, January 3, 1995; and in revised form, April 11, 1995)

Giovanna Allevato (1), Nils Billestrup (1)(§), Laure Goujon (2), Elisabeth D. Galsgaard (1), Gunnar Norstedt (3), Marie-Catherine Postel-Vinay (2), Paul A. Kelly (2), Jens H. Nielsen (1)

From the  (1)Hagedorn Research Institute, Niels Steensens Vej 6, DK 2820 Gentofte, Denmark, (2)Unité 344, Endocrinologie Moleculaire, Faculté de Médecine Necker, 75730 Paris Cedex 15, France, and the (3)Center for Biotechnology, Karolinska Institute, Novum, S 141 57 Huddinge, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The functional significance of growth hormone (GH) receptor (GHR) internalization is unknown; therefore, we have analyzed domains and individual amino acids in the cytoplasmic region of the rat GHR required for ligand-mediated receptor internalization, receptor down-regulation, and transcriptional signaling. When various mutated GHR cDNAs were transfected stably into Chinese hamster ovary cells or transiently into monkey kidney (COS-7) cells, internalization of the GHR was found to be dependent upon a domain located between amino acids 318 and 380. Mutational analysis of aromatic residues in this domain revealed that phenylalanine 346 is required for internalization. Receptor down-regulation in transiently transfected COS-7 cells was also dependent upon the phenylalanine 346 residue of the GHR, since no GH-induced down-regulation was observed in cells expressing the F346A GHR mutant. In contrast, the ability to stimulate transcription of the serine protease inhibitor 2.1 promoter by the GHR was not affected by the phenylalanine 346 to alanine mutation. These results demonstrate that phenylalanine 346 is essential for GHR internalization and down-regulation but not for transcriptional signaling, suggesting that ligand-mediated endocytosis is not a prerequisite for GH-induced gene transcription.


INTRODUCTION

The biological action of growth hormone (GH)()is initiated by its binding to specific receptors on the plasma membrane of target cells. The GH receptor (GHR) is a single trans-membrane protein consisting of an extracellular hormone binding domain with homology to other members of the cytokine/GH/prolactin receptor superfamily (1) and a cytoplasmic domain with little homology to the other members of the family and without any known catalytic activity. However, GH-induced tyrosine phosphorylation of the GHR has been demonstrated(2) , and this is probably dependent on the activation of a tyrosine kinase, JAK-2, which is associated with the receptor(3) . Recently it has been shown that one GH molecule binds to the extracellular region of two GHR molecules, indicating that receptor dimerization is necessary for biological activity(4) . Furthermore, receptor aggregation and endocytosis appear to play an essential role in transmembrane signaling of other growth factor receptors(5, 6) . Internalization itself has been suggested to be essential for signal transduction by the epidermal growth factor and other receptors(7, 8, 9) . The GHR is known to internalize in various cell types. In cultured rat adipocytes there appears to be constitutive internalization of the GHR, which is accelerated by the presence of GH(10) , whereas in IM-9 lymphocytes and mouse fibroblasts (11) the GHRs redistribute and aggregate in response to the addition of GH with no evidence for constitutive internalization. Interestingly, it has been suggested that part of the GHR itself exerts a biological effect after internalization based on the demonstration of GH binding protein and GHR in the nucleus of GH-stimulated cells(12) .

The domain(s) in the GHR involved in GH internalization has not yet been identified. In mutants of other receptors, those lacking the cytoplasmic domain accumulate at the cell surface, suggesting that signals required for rapid internalization are located in their cytoplasmic tails. Aromatic residues appear to be essential components of the internalization signals for most receptors(13) , yet the absence of any sequence similarities in the cytoplasmic region of receptors that are known to internalize rather suggests that it is a conformational signal for internalization that is recognized by a common cytoplasmic receptor(14) . Recent attempts to identify domains involved in the GHR signaling mechanism have shown that the conserved proline-rich box close to the trans-membrane region is essential for JAK-2 kinase activation (15) and mitogen-activated protein kinase activation as well as for the transcriptional activation of the serine protease inhibitor (SPI) 2.1 gene(16) . Similarly this region was also required for the mitogenic activity in GHR-transfected interleukin-3-dependent cells(17) . In addition to this region, the C-terminal 184 amino acids were required for expression of both the SPI 2.1 (16) and insulin (18) genes. Since the domain of the GHR required for internalization has not been identified, we have generated a series of mutated GHRs and tested their ability to internalize, down-regulate, and activate transcription after transfection.


EXPERIMENTAL PROCEDURES

Mutant Construction

The expression plasmid, pLM108, containing the full-length rat GH receptor cDNA under the transcriptional control of the human metallothionein IIa promoter and the simian virus 40 enhancer was constructed as described previously (19) . The cDNAs encoding GHR and GHR were generated from a BamHI/EcoRI fragment of the pLM108 plasmid, which was subcloned into M13 mp19 by primer-directed in vitro mutagenesis as described previously(18) . The remaining mutant cDNAs were constructed by using the polymerase chain reaction to splice out or alter regions as described(20) . Oligonucleotides carrying the different mutations were synthesized and used as primers in the polymerase chain reactions in order to introduce stop codons (GHR, GHR), deletions (GHR), and point mutations (GHR, GHR, GHR, and GHR) in the rat GHR cDNA. The introduced mutations were confirmed by DNA sequence analysis.

Cell Culture, Transfection, and Chloramphenicol Acetyltransferase (CAT) Assay

COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO in air. Cells were grown to 50% confluence in 100-mm cell culture dishes (Nunc, Roskilde, Denmark) and transiently transfected by a modified DEAE-dextran/chloroquine method (21, 22) with 5 µg of GHR expression plasmid containing the cDNA of either the wild type GHR or mutated GHR. Forty-eight hours after transfection the cells were tested for GH binding and internalization.

CHO K1 cells were cultured in Ham's F-12 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin at 37 °C in the presence of 5% CO. Cells were grown to 50% confluence in 60-mm dishes and were transiently transfected by the calcium phosphate procedure (23) with 3 µg of pCH110 (-galactosidase expression vector from Pharmacia Biotech Inc.), 1.5 µg of the construct containing the bacterial CAT coding sequence linked to the sequence -175/+59 of the serine protease inhibitor (SPI 2.1) promoter (24, 25) and 3 µg of the different mutated GHR plasmids. Cells were cultured for 48 h in the absence or presence of 400 ng/ml hGH, and cell extracts were normalized for -galactosidase activity and then assayed for CAT activity.

GH Receptor Binding, Down-regulation, and Internalization

COS-7 cells transiently transfected with the various GHR mutants were plated in 6-well dishes (Nunc) 24 h after transfection and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum for a further 24 h. The monolayers were carefully washed in Hepes binding buffer (10 mM Hepes, 124 mM NaCl, 4 mM CaCl, 1.5 mM MgCl, pH 7.4) and then incubated for 90 min at room temperature in binding medium (Hepes binding buffer, 1% human serum albumin) containing I-hGH (100,000 cpm). After incubation, the cells were washed four times in Hepes binding buffer and solubilized with 1 ml of 1 M NaOH. Radioactivity was counted in a -counter. Results are presented as the average of triplicate determinations from at least six experiments. Internalization and down-regulation were measured in COS-7 cells, which were plated in 6-well dishes and cultured as described above. For internalization measurements, monolayers were washed with Hepes binding buffer and kept on ice for 3 h in 1 ml of binding buffer containing 100,000 cpm I-hGH. Medium was then removed, and cells were rapidly warmed and incubated at 37 °C for the indicated time. Cells were again placed on ice, incubation medium was removed, and monolayers were washed four times with ice-cold Hepes binding buffer. Surface-associated (noninternalized) GH was determined by acid wash (0.15 M NaCl, 0.05 M glycine, pH 2.5)(26) , and internalized GH was determined by measuring the cell-associated radioactivity after solubilization with 1 M NaOH. Down-regulation was measured by incubating cells with 100 ng/ml of hGH for different time periods or overnight with different hGH concentrations. Free and surface bound hGH was removed by an acid wash, and residual GHRs at the cell surface were measured by I-hGH binding for 90 min at room temperature. After binding, the cells were washed four times in binding buffer, solubilized in 1 M NaOH, and counted in a -counter.


RESULTS

Growth Hormone Internalization in Cell Lines Expressing Truncated GH Receptors

A schematic representation of all the GHR mutants used in this study is shown in Fig. 1. A series of truncated GHRs were constructed by site-directed mutagenesis in order to identify regions in the intracellular part of the GHR involved in receptor internalization (Fig. 1, a-e). Deletion and point mutations were introduced in the conserved box 1 region, which previously has been shown to be required for JAK-2 binding and signal transduction (Fig. 1, f-g). Finally, aromatic residues in the region found to be required for internalization were mutated to alanines (Fig. 1, h-i). The binding of I-hGH to the various mutated GHRs was measured in COS-7 cells transiently transfected with each mutant GHR cDNA. All GHR constructs tested exhibited an IC of between 8.6 and 17.5 ng/ml of GH; no significant difference in IC between the various GHRs was observed, except GHR, which binds GH with a lowered affinity similar to that of the GH binding protein (27) (Fig. 2).


Figure 1: Schematic representation of the mutant GHR molecules. Mutants were constructed as described under ``Experimental Procedures.'' Signal peptide (SP), extracellular, transmembrane (TM), and intracellular domains are indicated by the hatched and shadedbars. The numbers correspond to amino acid residues. Shown are wild type (a), which consists of 638 amino acids including the putative signal peptide, mutant GHR (b), GHR (c), GHR (d), and GHR (e) in which lysine 455, alanine 381, lysine 319, or lysine 295, respectively, was mutated into a stop codon, GHR (f) in which 45 base pairs encoding amino acids 297-311 were deleted, GHR (P300,301,303,305A) (g) in which proline residues 300, 301, 303, and 305 were changed to alanine residues, GHR (YFY333,337,338A) (h) in which tyrosine residues 333 and 338 and phenylalanine residue 337 were changed to alanine residues, and mutant GHR (i), in which phenylalanine 346 was substituted with alanine.




Figure 2: GH binding competition curve in transiently transfected COS-7 cells. Binding of I-hGH to COS-7 cells transiently transfected with various GH receptor encoding plasmids is shown. COS-7 cells were cultured for 48 h after transfection, and binding of I-hGH in the presence of the indicated concentration of unlabeled hGH was measured. The IC values were calculated from the competition curves and represent the mean of four experiments ± S.D. GHR P300,301,303,305A, GHR; GHR YFY333,337,338A, GHR.



To examine the kinetics of GH uptake, we measured the internalization of GH in stably transfected CHO cells (data not shown) as well as in transiently transfected COS-7 cells. COS-7 cells expressing GHR, GHR, and GHR showed internalization of I-hGH, which reached a maximum after 1 h at 37 °C (Fig. 3). Approximately 60% of the specifically bound GH was internalized at this time point. As early as 15 min after raising the temperature to 37 °C, 25% of the specifically bound I-hGH was inside the cells. In contrast, in COS-7 cells expressing GHR internalization was greatly reduced, with only 15% of the specifically bound GH internalized after 1 h. GHR showed approximately the same internalization rate as GHR. These data indicate that a domain required for internalization of the GHR is located between residues 318 and 380. Similar results were observed in stably transfected CHO cells (data not shown).


Figure 3: Internalization of surface-bound GH by COS-7 cells expressing wild type and mutant GHRs. Cells were incubated as described under ``Experimental Procedures.'' At various time points, the cells were washed with acidic buffer and solubilized to determine intracellular radioactivity. Each point represents the mean of duplicate measurements in 5-10 experiments. The S.D. values were <3%. Values are expressed as the percentage of initial bound counts at time 0 and corrected for nonspecific internalization at each time point. GHR P300,301,303,305A, GHR; GHR YFY333,337, 338A, GHR.



Localization of the Amino Acid Residues Involved in GHR Internalization

To evaluate the contribution of the proline-rich region in ligand-mediated endocytosis we constructed two GHR mutants: one with this portion deleted, GHR, and the other in which the four proline residues present in this region were changed into alanines, GHR (Fig. 1). Plasmids encoding either mutated receptor were transfected into CHO and COS-7 cells. As shown in Fig. 3, GHR demonstrated internalization of GH in COS cells similar to the wild-type GHR, whereas internalization was reduced in cells expressing GHR. In view of the identification in many other transmembrane receptors of potential internalization domains in which aromatic residues are important, we examined the role of aromatic residues present in the cytoplasmic domain of the GHR. In the region between 318 and 380 only four aromatic residues are present: tyrosine 333, phenylalanine 337, tyrosine 338, and phenylalanine 346. We therefore constructed a GHR mutant in which tyrosines 333 and 338 and phenylalanine 337 were mutated into alanines, GHR, and a mutant in which phenylalanine 346 was mutated into alanine, GHR (Fig. 1). As shown in Fig. 3, GHR internalized GH as effectively as the wild type receptor. In contrast, GHR only internalized to the same extent as mutant GHR, which lacks all but five amino acids of the cytoplasmic region.

Role of Phenylalanine 346 in GHR Down-regulation

In order to compare the domain of the GHR required for internalization with the domain required for down-regulation of the GHR, we examined GH-induced GHR down-regulation in transiently transfected COS-7 cells. Cells expressing the wild-type GHR showed a dose- and time-dependent down-regulation of the GHR (Fig. 4). After a 5-min exposure to 100 ng/ml GH, 60% of specific GHRs were down-regulated, and after 10 min 80% of GHRs were down-regulated (Fig. 4A). This level of down-regulation remained constant for up to 18 h. In contrast, COS-7 cells expressing either the GHR or the GHR mutant did not exhibit GH-induced receptor down-regulation. An initial reduction in binding of about 25% was observed after a 5-10-min exposure to 100 ng/ml of GH; however, this apparent down-regulation was transient since binding returned to control levels after 60 min of exposure to GH. This rapid loss of GH binding sites observed in cells expressing the GHR or the GHR mutant might be caused by membrane turnover, and longer exposure to GH might allow for the insertion of newly synthesized GHRs into the plasma membrane. A dose-dependent down-regulation of the wild-type GHR but not of the mutated GHRs was observed after an 18-h exposure to GH (Fig. 4B). These data show that phenylalanine 346 is required for both internalization and down-regulation and suggests that receptor internalization is the cause of down-regulation.


Figure 4: Down-regulation of GHRs in COS-7 cells expressing wild-type and mutated GHRs. Transiently transfected COS-7 cells were incubated with 100 ng/ml of hGH for various time points (A) or overnight with the indicated concentration of hGH (B), and GHR down-regulation was measured as described under ``Experimental Procedures.'' Data from cells expressing the wild-type GHR (), GHR () and GHR () are shown. Each point represents the mean of duplicate measurements from three experiments.



Role of GH Internalization in Signal Transduction

The biological activity of the GHR mutants was evaluated by a transient co-transfection assay in which the ability of GH to stimulate the expression of the SPI 2.1 promoter/CAT gene construct in CHO cells was tested. As shown in Fig. 5, deletion of only 184 amino acid residues in the C-terminal region of the GHR resulted in loss of this activity. Furthermore, deletion of the proline-rich box(297-311) or mutation of the four proline residues (300, 301, 303 and 305) to alanines abolished transcriptional activity as described previously (16) . In contrast, mutation of the aromatic residues 333, 337, and 338 to alanine did not cause any reduction in activity. In addition, substitution of phenylalanine 346 with alanine in the GHR, despite being deficient in internalization and down-regulation, was fully capable of stimulating SPI 2.1 promoter transcriptional activity.


Figure 5: Transcriptional activity of mutant GHRs in the SPI/CAT assay. CHO cells were transiently co-transfected with plasmids containing cDNAs encoding either the wild type (GHR) or the indicated mutated GHR cDNAs together with the fusion gene SPI/CAT. After transfection the cells were incubated in the presence or absence of 400 ng/ml hGH 48 h. The -fold induction was calculated as percentage of chloramphenicol conversion in the presence of GH divided by the percentage of conversion in the absence of GH. Results represent the mean ± S.D. of 5-10 independent experiments. GHR P300,301,303,305A, GHR; GHR YFY333,337,338A, GHR.




DISCUSSION

The present results indicate that internalization and transcriptional signaling by the GHR are independent events. Results obtained using CHO (data not shown) and COS-7 cells confirm and extend our previous data from RIN cells(18) , which demonstrated that a C-terminal truncation of the GHR at position 454 results in loss of GH-stimulated insulin expression but not the ability to internalize. In addition, truncation at position 294 resulted in loss of both activities. These data indicate that the domain required for internalization is located between residues 295 and 455 and that receptor internalization itself cannot initiate GH actions. The paradoxical finding that deletion of box 1 had no effect on internalization whereas mutation of the four proline residues reduced internalization suggests that the profound distortion of the conformation by changing all four proline residues prevents the domain required for internalization from interacting with the putative cytoplasmic receptor recognizing internalization signals(14) . Since the GHR was able to internalize to the same extent as the full-length GH receptor, while GHR or GHR exhibited impaired internalization, we concluded that the domain required for internalization was located between amino acid residues 318 and 380. In this region only four aromatic residues are present; therefore, we analyzed their role in internalization. From the present study it appears that an aromatic residue at position 346, but not in position 333, 337, or 338, is essential for internalization, since the substitution of phenylalanine 346 with alanine inhibited internalization but not transcriptional activity. Whether the phenylalanine 346 is directly involved in the internalization process or it is simply required for the overall conformation of a region involved in internalization cannot be determined by this study. It is, however, unlikely that the tertiary structure of the GHR is changed dramatically by the alanine substitution, since the transcriptional activation by the GHR mutant is unchanged. Furthermore alanine substitutions eliminate side chains beyond the -carbon and do not impose steric or electrostatic effects. Substitutions by alanine have been used frequently by others and shown not to change the tertiary structure of many proteins(28, 29, 30) .

Aromatic residues have been shown to be important for the internalization of other receptors. For the transferrin receptor, the internalization sequence is YXRF(31) , and for the LDL and insulin receptors it is NPXY(32, 33) ; both these sequences are localized in the cytoplasmic domain close to the transmembrane region, but except for the aromatic residue, there are no other additional sequence similarities. Conformational studies have led to the conclusion that an aromatic residue present in the context of a tight turn appears to be a common structural motif necessary for internalization signals(34, 35, 36) . Our results fit this general concept and further support a direct role of phenylalanine 346 as being important for the internalization sequence in the GHR.

Other biological effects of GH also do not require the C-terminal domain of the receptor, as demonstrated in recent studies where GHR, although unable to stimulate SPI 2.1 transcription, was still found to be able to transmit the signal for protein biosynthesis, mitogen-activated protein kinase, and JAK-2 kinase activation and mitotic activity in CHO cells(15, 37) . Furthermore, in the interleukin-3-dependent promyeloid cell line FDC-P1, the human GHR truncated at position 325 (corresponding to 344 in the present numbering of the rat GHR) was able to transmit a mitogenic signal, indicating that this activity exclusively resides in box 1, although the effect was augmented when box 2 was retained(17) . Since both GH and several other growth factors and cytokines are reported to activate JAK-2 kinase (38, 39) it may be speculated that biological responses common to these growth factors such as protein biosynthesis, mitogen-activated protein kinase activation, and mitotic activity are due to binding and activation of JAK-2 kinase by box 1, which then interacts with box 2 in a yet unknown manner. However, activities attributed to specific members of this receptor family rely on domains in the C-terminal region. Since nuclear localization of both GH and GHR has been demonstrated, it has been proposed that GH, GHR, or GH binding protein exerts biological functions after being internalized (12) . The present finding of a GHR mutant that is not internalized but retains full transcriptional activity of the SPI 2.1 promoter indicates, however, that such mechanisms are probably not involved in this activity, but GHR internalization might be required for other biological actions. In conclusion, these results support the hypothesis that different domains in the cytoplasmic region of the GHR are responsible for transducing signals for separate biological activities of GH.


FOOTNOTES

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

§
To whom correspondence should be addressed: Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark. Tel.: 45 31 68 08 60; Fax: 45 31 68 05 02; nbil{at}hrl.dk

The abbreviations used are: GH, growth hormone; GHR, GH receptor; SPI, serine protease inhibitor; CAT, chloramphenicol acetyltransferase; CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We thank Dr. Povl Nilsson and Charlotte Bj(Novo Nordisk A/S, Gentofte, Denmark) for the preparation of iodinated hGH. We thank Tina Kisbye and Jannie Rosendahl Christensen for excellent technical assistance. We thank Drs. Erica Nishimura and Annette MPerregaard for critical review of the manuscript.


REFERENCES
  1. Cosman, D., Lyman, S., Idzerda, D. R. L., Beckmann, M. P., Park, L. S., Goodwin, R. G., and March, C. J.(1990)Trends Biochem. Sci. 15, 265-270 [CrossRef][Medline] [Order article via Infotrieve]
  2. Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C.(1988)Biochemistry 27, 326-334 [Medline] [Order article via Infotrieve]
  3. Argetsinger, L. S., Campbell, G. S., Wang, X., Witthuhn, B. A., Silvennoinen, O., Ihle, J. N., and Carter-Su, C.(1993)Cell 74, 237-244 [Medline] [Order article via Infotrieve]
  4. Fuh, G., Cunningham, B. C., Fukunaga, R., Nagata, S., Goeddel, D. V., and Wells, J. A. (1992)Science 256, 1677-1680 [Medline] [Order article via Infotrieve]
  5. Ullrich, A., and Schlessinger, J.(1990)Cell 61, 203-12 [Medline] [Order article via Infotrieve]
  6. Schlessinger, J., and Ullrich, A.(1992)Neuron 9, 383-91 [Medline] [Order article via Infotrieve]
  7. Savion, N., Vlodavsky, I., and Gospodarowicz, D.(1981)J. Biol. Chem. 256, 1149-1154 [Abstract/Free Full Text]
  8. Wakshull, E. M., and Wharton, W.(1985)Proc. Natl. Acad. Sci. U. S. A. 82, 8513-8517 [Abstract]
  9. Yanker, B. A., and Shooter, E. M.(1982)Annu. Rev. Biochem. 51, 845-868 [Medline] [Order article via Infotrieve]
  10. Roupas, P., and Herington, A.(1988)Mol. Cell. Endocrinol. 57, 93-99 [CrossRef][Medline] [Order article via Infotrieve]
  11. Eshet, R., Peleg, S., and Laron, Z.(1984)Acta Endocrinol. 107, 9-15 [Medline] [Order article via Infotrieve]
  12. Lobie, P. E., Barnard, R., and Waters, M. J.(1991)J. Biol. Chem. 266, 22645-22652 [Abstract/Free Full Text]
  13. Trowbridge, I. S. (1991)Curr. Opin. Cell Biol. 3, 634-641 [Medline] [Order article via Infotrieve]
  14. Pearse, B. M. F., and Robinson, M. S.(1990)Annu. Rev. Cell Biol. 6, 151-171 [CrossRef]
  15. VanderKuur, J. A., Wang, X., Zhang, L., Campbell, G. S., Allevato, G., Billestrup, N., Norstedt, G., and Carter-Su, C.(1994)J. Biol. Chem. 269, 21709-21717 [Abstract/Free Full Text]
  16. Goujon, L., Allevato, G., Simonin, G., Paquereau, L., Le Cam, A., Clarks, J., Nielsen, J. H., Djiane, J.-M., Postel-Vinay, M.-C., Edery, M., and Kelly, P. A. (1994) Proc. Natl. Acad. Sci. U. S. A.91, 957-961 [Abstract]
  17. Colosi, P., Wong, K., Leong, S. R., and Wood, W. I.(1993)J. Biol. Chem. 268, 12617-12623 [Abstract/Free Full Text]
  18. M, A., Allevato, G., Dyrberg, T., Nielsen, J. H., and Billestrup, N. (1991)J. Biol. Chem. 266, 17441-17445 [Abstract/Free Full Text]
  19. Billestrup, N., Mldrup, A., Serup, P., Mathews, L. S., Norstedt, G., and Nielsen, J. H. (1990)Proc. Natl. Acad. Sci. U. S. A. 87, 7210-7214 [Abstract]
  20. Herlitze, S., and Koenen, M.(1990)Gene (Amst.) 91,143-147 [Medline] [Order article via Infotrieve]
  21. McCutchan, J. H., and Pagano, J. S.(1968)J. Natl. Cancer Inst. 41, 351-357 [Medline] [Order article via Infotrieve]
  22. Luthman, H., and Magnum, G.(1983)Nucleic Acids. Res. 11, 1295-1308 [Abstract]
  23. Southern, P. J., and Berg, P.(1982)J. Mol. Appl. Genet. 1, 327-341 [Medline] [Order article via Infotrieve]
  24. Yoon, J. B., Berry, S. A., Seelig, S., and Towle, H.(1990)J. Biol. Chem. 265, 19947-19954 [Abstract/Free Full Text]
  25. Paquereau, L., Vilarem, M. J., Rossi, V., Rouayrenc, J. F., and Le Cam, A.(1992) Eur. J. Biochem. 209, 1053-1061 [Abstract]
  26. Roupas, P., and Herington, A. C.(1986)Mol. Cell. Endocrinol. 47, 81-90 [CrossRef][Medline] [Order article via Infotrieve]
  27. Leung, D. W., Spencer, S. A., Cachianes, G., Hammonds, R. G., Collins, C., Henzel, W. J., Barnard, R., Waters, M. J., and Wood, W. I.(1987) Nature 330, 537-543 [CrossRef][Medline] [Order article via Infotrieve]
  28. Cunningham, B. C., and Wells, J. A.(1989)Science 244, 1081-1085 [Medline] [Order article via Infotrieve]
  29. Bass, S. H., Mulkerrin, M. G., and Wells, J. A.(1991)Proc. Natl. Acad. Sci. U. S. A. 88, 4498-4502 [Abstract]
  30. Jin, L., Fendly, B. M., and Wells, J. A.(1992)J. Mol. Biol. 226, 851-865 [Medline] [Order article via Infotrieve]
  31. Jing, S., Spencer, T., Miller, K., Hopkins, C., and Trowbridge, I. S.(1990) J. Cell Biol. 110, 283-294 [Abstract]
  32. Chen, W.-J., Goldstein, J. L., and Brown, M. S.(1990)J. Biol. Chem. 265, 3116-3123 [Abstract/Free Full Text]
  33. Backer, J. M., Kahn, C. R., Cahill, D. A., Ullrich, A., and White, M.(1990) J. Biol. Chem. 265, 16450-16454 [Abstract/Free Full Text]
  34. Eberle, W., Sander, C., Klaus, W., Schmidt, B., von Figura, K., and Peters, C.(1991) Cell 67, 1203-1209 [Medline] [Order article via Infotrieve]
  35. Collawan, J. F., Stangel, M., Khun, L. A., Esekogwu, V., Jing, S., Trowbridge, I. S., and Tainer, J.(1990)Cell 63, 1061-1072 [Medline] [Order article via Infotrieve]
  36. Bansal, A., and Gierash, L. M.(1991)Cell 67, 1195-1201 [Medline] [Order article via Infotrieve]
  37. Möller, C., Hansson, A., Enberg, B., Lobie, P. E., and Norstedt, G.(1992) J. Biol. Chem. 267, 23403-23408 [Abstract/Free Full Text]
  38. Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Yi, T., Tang, B., Miura, O., and Ihle, J. N. (1993)Cell 74, 227-36 [Medline] [Order article via Infotrieve]
  39. Watling, D., Guschin, D., Muller, M., Silvennoinen, O., Witthuhn, B. A., Quelle, F. W., Rogers, N. C., Schindler, C., Stark, G. R., Ihle, J. N., and Kerr, I. M.(1993)Nature 366, 166-170 [CrossRef][Medline] [Order article via Infotrieve]

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