| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
1Department of Endocrinology, University of Pisa, Pisa, Italy; 2Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; 3Departments of Medicine and Pediatrics, University of Chicago, Chicago, Illinois; 4Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, Michigan; 5Departments of Pediatrics, Cell Biology, and Physiology, Washington University School of Medicine, St. Louis, Missouri; and 6Program in Membrane Biology, Renal Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts
Submitted 29 July 2005 ; accepted in final form 16 November 2005
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
low-density lipoprotein; knockout mice
39-kDa endoplasmic reticulum (ER)-resident protein that functions as a molecular chaperone for several members of the LDL receptor family, including megalin (6, 23). RAP is present in virtually all cell types that express megalin, where it is localized in the ER and early Golgi compartments via a specialized COOH-terminal ER retention signal (6, 23). In the absence of RAP, folding of megalin is impaired, and as a result, the receptor is retained within the ER with reduced expression on the cell membrane (4, 6, 23–25). Both megalin and RAP are expressed by thyroid epithelial cells (thyrocytes) (26), where megalin functions as an endocytic receptor for the thyroid hormone precursor thyroglobulin (Tg). Tg is synthesized by thyrocytes and then secreted into the lumina of thyroid follicles, where, in the proximity of the thyrocyte apical membrane, it undergoes hormone formation by the coupling of its tyrosyl residues with iodine (1). Subsequently, hormone-rich Tg molecules can either proceed further into the follicle, where they arrange into aggregates that form the colloid, or can undergo proteolytic cleavage, ultimately resulting in hormone release (1). The latter process occurs mainly in lysosomes after fluid-phase micropinocytosis of Tg by thyrocytes (1), as well as in the lumina of thyroid follicles through the action of extracellular proteases (5, 7). Megalin is expressed on the apical membrane of thyrocytes (26), where it binds to Tg with high affinity, after which Tg is internalized by cells to undergo apicobasolateral transcytosis and is released into the bloodstream (14, 17). The megalin-mediated transcytosis pathway preferentially involves hormone-poor Tg molecules, which, by preventing lysosome engulfment as well as wasteful transcytosis of hormone-rich Tg, renders hormone release more effective (14).
The absence of RAP in thyrocytes should affect megalin expression and function, which might result in a thyroid phenotype similar to that observed in megalin-knockout (megalin-KO) mice, namely, hypothyroidism (14, 15). However, the effects of RAP deficiency may be more complex, because RAP interacts not only with megalin but also with Tg (16). Thus we have recently demonstrated (16) that RAP binds to Tg in solid-phase assays with relatively high affinity and that antibodies against RAP can coprecipitate Tg in thyroid extracts and in cultured thyroid cells; in the latter case, this phenomenon occurs shortly after the beginning of Tg biosynthesis. In addition, transient transfection of differentiated thyroid cells with a secretory RAP-Ig chimera devoid of the ER retention sequence results in impaired Tg release into the cell medium, suggesting that RAP may be involved in the Tg biosynthetic pathway (13). Nevertheless, it is not known how the role of RAP in Tg biosynthesis might affect thyroid function in combination with its role in megalin expression.
To elucidate the function of RAP in thyrocytes in the present study, we investigated and characterized the thyroid phenotype in RAP-KO mice. Our findings indicate that in the absence of RAP, a defect in follicular Tg associated with slight histological changes resembling early goiter occurs. Hormone secretion was normal, indicating that follicular Tg was still sufficient and sufficiently iodinated to provide hormone secretion.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Blood samples were collected from 15 RAP-KO (10 female and 5 male) and 15 WT mice (10 female and 5 male). Sera were stored at –20°C until use. The thyroid glands were harvested in all mice and either stored at –80°C for preparation of tissue extracts or fixed with formalin and embedded in paraffin for histological and immunohistochemical studies.
Immunohistochemistry. Four- to five-µm-thick sections from paraffin-embedded specimens were deparaffinized, rehydrated, and subjected to antigen retrieval either in Citra, pH 6.0 (BioGenex, San Ramon, CA), for RAP and Tg detection, or in Tris buffer, pH 10.0, for megalin detection using a decloaking chamber (Biocare Medical, Walnut Creek, CA). Sections were then washed with PBS and incubated with rabbit antibodies against RAP (26), megalin (26), or Tg (Axell, Westbury, NY), followed by biotin-labeled horse anti-rabbit IgG (Vector Laboratories, Burlingame, CA), horseradish peroxidase (HRP)-conjugated streptavidin (Elite ABC reagent; Vector Laboratories), and 3-amino-9-ethylcarbazole (Aldrich Chemical, Milwaukee, WI). Sections were counterstained with Gill's hematoxylin (Fisher Scientific, Fair Lawn, NJ).
Tissue extract preparation and analysis. Tissue samples were washed, minced, and homogenized. Samples were then incubated in lysis buffer [50 mM Tris, pH 8.6, 0.5 M NaCl, 10% Triton X-100, 0.01% deoxycholate, 20 mM EDTA, 0.2% NaN3, and 10% protease inhibitor cocktail solution (Roche Diagnostics, Mannheim, Germany)] and spun for 30 min at 10,000 g. Supernatants were collected and briefly sonicated. To measure poorly soluble Tg aggregates within the pellets, the pellets were collected and subjected to further solubilization by incubation with 6 M guanidine.
Protein concentrations were measured in all samples using the Bradford method. Tg was measured using either Western blot analysis as detailed below or ELISA as follows. Microwell plates coated with the rabbit anti-Tg antibody were incubated with 1 µg of extracts, followed by an unlabeled mouse anti-Tg antibody (Dako, Carpinteria, CA), alkaline phosphatase-conjugated anti-mouse IgG (Sigma, St. Louis, MO) and para-nitrophenylphosphate (Sigma). Tg values were calculated on the basis of a standard curve obtained using mouse Tg prepared from frozen mouse thyroids (Pel-Freeze, Rogers, AR).
Total thyroxine (T4) was measured in thyroid extracts diluted in T4-, T3-free human serum using a commercially available ELISA kit (MP Biomedicals, Orangeburg, NY). T4 residues within Tg molecules were measured by ELISA as follows. Microtiter plates coated with unlabeled rabbit anti-Tg were incubated with thyroid extracts (1 µg), followed by a rabbit anti-T4 antibody (Accurate Chemical and Scientific, Westbury, NY) labeled with N-sulfosuccinimidyl-6-[biotinamido]hexanoate (EZ-Link sulfo-NHS-LC-biotin; Pierce Biotechnology, Rockford, IL) and then by alkaline phosphatase-conjugated streptavidin and para-nitrophenylphosphate. The amount of T4 within Tg was expressed as optical density/ng Tg as measured by ELISA in the same samples.
Western blot analysis. Samples were subjected to SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with 1) unlabeled rabbit anti-megalin or anti-Tg antibodies, followed by HRP-conjugated anti-rabbit IgG (Bio-Rad Laboratories, Hercules, CA); 2) unlabeled mouse anti-Tg antibody, followed by HRP-conjugated anti-mouse IgG (Bio-Rad Laboratories); or 3) HRP-conjugated mouse anti-Tg antibody (Dako). Bands were detected using ECL, and their density was measured using ImageJ software (National Institutes of Health, Bethesda, MD).
Serum assays. Free T4 (FT4) was measured by performing an equilibrium dialysis immunoassay (Nichols, San Juan Capistrano, CA). TSH was measured using a sensitive, heterologous disequilibrium double-antibody precipitation RIA (21). Serum Tg was measured by ELISA as follows. Microwell plates coated with the rabbit anti-Tg antibody were incubated with biotin-labeled mouse Tg alone or in the presence of mouse sera diluted 1:25, followed by alkaline phosphatase-conjugated streptavidin and para-nitrophenylphosphate. Tg concentrations were calculated on the basis of a lin/log, competitive, RIA-like curve obtained by incubating anti-Tg antibody-coated wells with biotin-labeled Tg in the presence of various concentrations of unlabeled mouse Tg. The arbitrary value of 1 U/ml was assigned to the result obtained with 1 µg/ml unlabeled Tg.
Data presentation and statistics. Experiments with mouse tissue sections and extracts were performed in at least four mouse pairs. Serum assays were performed in 15 mouse pairs (5 male and 10 female), with the exception of serum TSH, which was measured only in females because TSH values are known to differ between the genders in mice (21) and not enough serum samples were available in males. Serum TSH values in RAP-KO and WT mice were also compared with those obtained in 16 serum samples from a separate series of WT mice (WT2). This additional group of serum samples was selected from our archives and included all samples from mice that shared the same genetic background (B6), gender, and age as the RAP-KO mice and the mice in the first WT group. These mice had been maintained under the same care conditions as the RAP-KO mice and the mice in the first WT group. In all experiments, either the same sample volumes (serum) or the same amount of protein (extracts) were used for comparison. When appropriate, statistical analyses were performed using an unpaired t-test or simple regression with StatView software (SAS Institute, Cary, NC). Results are presented as means ± SD.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
As expected on the basis of the knowledge that RAP is an ER-resident protein (6, 25), RAP was found intracellularly by immunohistochemistry in thyrocytes of WT mice (Fig. 1A). RAP was not detected in thyroid sections from RAP-KO mice, confirming that the RAP gene had been disrupted successfully (Fig. 1B).
|
Reduced follicular Tg in RAP-KO mice. Further immunohistochemical studies in which we used an anti-Tg antibody revealed a reduction of Tg in thyroid follicles in RAP-KO mice. As shown in Fig. 2A, in WT mice, abundant staining for Tg was found in the lumina of thyroid follicles, together with some intracellular staining. In contrast, in RAP-KO mice, Tg staining was increased intracellularly, whereas in the lumina of the vast majority of follicles, staining was either undetectable or markedly reduced (Fig. 2B). In RAP-KO mice, intense Tg staining also was observed on the apical membrane of thyrocytes, which was not clearly detectable in WT mice.
|
25% lower in RAP-KO mice, the difference was not statistically significant (P = 0.4275; t-test). Western blot analysis revealed that 660- and 330-kDa Tg bands (corresponding to intact Tg dimers and monomers) in RAP-KO mice were reduced by only 20% and 15%, respectively (mean densities: 660-kDa Tg, 39.1 pixels/cm2 vs. 48.1 pixels/cm2 in WT mice; 330-kDa Tg, 39.4 pixels/cm2 vs. 45.7 pixels/cm2 in WT mice). Moreover, an additional Tg band migrating at 200 kDa, presumably representing a proteolytic fragment, was reduced by only 20% in RAP-KO mice (16.5 vs. 20.9 pixels/cm2 in WT mice). Because of the apparent discrepancy between results obtained with thyroid sections and those obtained with thyroid extracts, we considered the possibility that the reduction of Tg in RAP-KO mice observed using immunohistochemistry was due to a reduction of Tg aggregates that may not have been solubilized by standard tissue extraction. Thus follicular Tg can be stored in the form of aggregates (11) that may have been discarded during the extraction procedure, because they are expected to remain within the pellets upon centrifugation of homogenates. To investigate this possibility, the pellets from thyroid homogenates were collected, subjected to solubilization by treatment with 6 M guanidine, and examined using Western blot analysis. As shown in Fig. 2E, 660- and 330-kDa Tg bands were detected in the material solubilized from the thyroids of WT mice but not in that from the thyroids of RAP-KO mice, supporting the conclusion that the reduction of follicular Tg in RAP-KO mice reflects a reduction of Tg aggregates that were not solubilized by the standard extraction procedure.
Tg in RAP-KO mice undergoes hormone formation as it does in WT mice. We investigated whether the few Tg molecules found in the colloid in RAP-KO mice, especially those bound to the apical membrane (Fig. 2B), could still undergo hormone formation. For this purpose, we first measured the content of total T4 in thyroid extracts, which was found to be reduced only slightly in RAP-KO mice (Fig. 3A), with a difference with WT mice that was not statistically significant (P = 0.4034; t-test) and with values that paralleled those of Tg in the same extracts (Fig. 2C). Thus there was a significant correlation between Tg and total T4 in thyroid extracts from both WT (P = 0.0483; simple regression analysis) and RAP-KO mice (P = 0.0440) (Fig. 3B). The findings indicate that Tg in RAP-KO mice could still undergo hormone formation. In further support of these findings, the content of T4 within Tg molecules in thyroid extracts did not differ significantly (P = 0.4340; t-test) between WT and RAP-KO mice, and interestingly, mean values were slightly greater in RAP-KO mice (Fig. 3C). We interpreted the latter finding as possibly due to a likely increased iodine-to-Tg ratio within the colloid in RAP-KO mice, which was in fact expected in view of the reduction of follicular Tg.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
The major conclusion of the present study is that RAP is required for the establishment of large reservoirs of Tg within thyroid follicles. Thus mice generated by disruption of the RAP gene were found to have a marked reduction of Tg content in thyroid follicles, whereas much of the Tg synthesized by thyrocytes was retained intracellularly or secreted into the bloodstream. The reduction of follicular Tg in RAP-KO mice reflected especially a reduction of poorly soluble Tg aggregates, one of the forms in which Tg accumulates as an iodine and hormone reservoir (1, 11).
RAP-KO mice had significantly higher serum levels of Tg, suggesting that there may have been a directional alteration of Tg secretion, namely, that Tg was secreted from basolateral membranes into the bloodstream rather than from apical membranes into follicles. Other mechanisms through which Tg can reach the bloodstream, namely, transcytosis or leakage from follicles (10), are unlikely to account for the elevated serum Tg levels. Thus megalin-mediated transcytosis, the main route of Tg transcytosis (14, 15, 17), could not be increased, because megalin on the apical membrane of thyrocytes was reduced. In addition, leakage from follicles could be excluded because of the absence of histological evidence of follicle disruption.
In spite of the severe defect of follicular Tg, thyroid function was maintained in RAP-KO mice. Several findings and considerations may explain why RAP-KO mice did not develop hypothyroidism. First, the presence of T4 in thyroid extracts, the finding that its levels paralleled those of Tg, and more important, the finding that Tg contained T4 residues indicate that the few Tg molecules secreted into the colloid had undergone hormone formation. Second, Tg within follicles in RAP-KO mice was observed almost exclusively near the apical membrane of thyrocytes, hence in the ideal position for being iodinated rapidly, internalized, and degraded. Thus it is likely that, although not enough Tg was present in the follicles to form large reservoirs, its quantities were sufficient to provide normal hormone secretion. This interpretation is in keeping with the so-called last come, first served hypothesis (22). According to this notion, under normal conditions, the Tg used for hormonogenesis is the last to be secreted into the colloid. These newly secreted molecules would remain near the apical membrane of thyrocytes for a short time, where they would undergo hormone formation and be internalized and/or degraded rapidly. In contrast, the Tg in the center of the follicle would be stored in the form of aggregates serving as an iodine and hormone reservoir, to be used only when the iodine supply is not sufficient for newly secreted Tg to undergo hormone formation. As mentioned above, in RAP-KO mice there was a reduction of Tg aggregates, but Tg secretion probably was sufficient for hormone release.
Serum TSH was increased in RAP-KO mice, but the difference from that of WT mice was not statistically significant. However, when we compared serum TSH values with those of a larger archival series of WT mice (WT2) of the same genetic background, gender, and age, TSH was found to be significantly greater in RAP-KO mice. In fact, certain histological features (thyrocyte hypertrophy and nuclear enlargement) known to be triggered by TSH and to resemble early stages of goiter (3) were observed in RAP-KO mice, which is in keeping with increased TSH secretion. Other features of TSH stimulation, namely, thyroid enlargement, hypercellularity, increased follicle area, and hypervascularity were not observed. Hence, TSH secretion, although increased, probably still was not sufficient to trigger a full-blown goitrous phenotype.
In addition to the defect of follicular Tg, RAP-KO mice also showed a reduction in megalin on the apical membrane of thyrocytes, presumably because of the role of RAP as a megalin chaperone (6, 25). Because megalin mediates the transcytosis of Tg with subsequent release of Tg into the bloodstream (14, 17), serum Tg would be expected to be reduced in RAP-KO mice just as it occurs in megalin-KO mice (14, 15). However, RAP-KO mice had increased serum levels of Tg. How could this finding be coupled with the reduction of megalin? An important issue to consider is that although transcytosis is one of the mechanisms by which Tg can reach the circulation, it is not the only one, as mentioned above. Thus Tg also can enter the bloodstream by direct cell secretion or by leakage from disrupted follicles (10). A reduction in transcytosis should cause a decrease in serum Tg only if these two additional mechanisms are not increased. This is the case in megalin-KO mice, in which follicular Tg is normal and there is no follicle disruption (15), but probably is not the case in RAP-KO mice, in which, although there was no follicle disruption, directional secretion of Tg was likely altered. If this were indeed the case, the increased serum Tg might have reflected enhanced basolateral secretion and might have masked reduced transcytosis.
Another important question concerning megalin is why RAP-KO mice did not develop hypothyroidism in spite of the reduced megalin expression. Thus, by mediating transcytosis of hormone-poor Tg, megalin prevents it from competing with hormone-rich Tg for proteolytic degradation, and as a consequence, megalin-KO mice have hypothyroidism (14, 15). A possible explanation for this apparent discrepancy is related to the hormone content of Tg. Transcytosis should have an impact on thyroid function only when there is enough hormone-poor Tg within the colloid (14), which probably was not the case in RAP-KO mice. This hypothesis is supported by the fact that Tg in thyroid extracts from RAP-KO mice contained T4 as in WT mice and, more important, by the finding that the T4 content of Tg in RAP-KO mice was slightly greater than that in WT mice, although not to a statistically significant extent. Because extracts comprised both intracellular and colloidal Tg, intracellular Tg was increased in RAP-KO mice, and intracellular Tg is known not to be iodinated, the hormone content of colloidal Tg in RAP-KO mice might have been even greater than that which we measured. Moreover, in RAP-KO mice, the iodine-to-Tg ratio within the colloid was probably increased because of the reduction in follicular Tg, and therefore an enhanced hormone content of Tg was likely to be present. On the basis of these considerations, it is possible that megalin did not affect thyroid function in RAP-KO mice simply because the few Tg molecules within the colloid had a high hormone content. Under the circumstances, transcytosis of hormone-poor Tg probably was not only not possible but also not necessary. A tentative, schematic view of how the absence of megalin or RAP might affect Tg trafficking and thyroid function in the thyrocyte is depicted in Fig. 5.
|
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Arvan P, Kim PS, Kuliawat R, Prabakaran D, Muresan Z, Yoo SE, and Abu Hossain S. Intracellular protein transport to the thyrocyte plasma membrane: potential implications for thyroid physiology. Thyroid 7: 89–105, 1997.[ISI][Medline]
3. Baloch ZA and Livolsi VA. Pathology. In: Werner and Ingbar's The Thyroid: A Fundamental and Clinical Text (9th ed.), edited by Braverman LE and Utiger RD. Philadelphia, PA: Lippincott Williams & Wilkins, 2005, p. 422–449.
4. Birn H, Vorum H, Verroust PJ, Moestrup SK, and Christensen EI. Receptor-associated protein is important for normal processing of megalin in kidney proximal tubules. J Am Soc Nephrol 11: 191–202, 2000.
5. Brix K, Linke M, Tepel C, and Herzog V. Cysteine proteinases mediate extracellular prohormone processing in the thyroid. Biol Chem 382: 717–725, 2001.[CrossRef][ISI][Medline]
6. Bu G. The roles of receptor-associated protein (RAP) as a molecular chaperone for members of the LDL receptor family. Int Rev Cytol 209: 79–116, 2001.[ISI][Medline]
7. Delom F, Mallet B, Carayon P, and Lejeune PJ. Role of extracellular molecular chaperones in the folding of oxidized proteins: refolding of colloidal thyroglobulin by protein disulfide isomerase and immunoglobulin heavy chain-binding protein. J Biol Chem 276: 21337–21342, 2001.
8. De Vijlder JJM. Primary congenital hypothyroidism: defects in iodine pathways. Eur J Endocrinol 149: 247–256, 2003.[Abstract]
9. Di Jeso B, Ulianich L, Pacifico F, Leonardi A, Vito P, Consiglio E, Formisano S, and Arvan P. Folding of thyroglobulin in the calnexin/calreticulin pathway and its alteration by loss of Ca2+ from the endoplasmic reticulum. Biochem J 370: 449–458, 2003.[CrossRef][ISI][Medline]
10. Druetta L, Bornet H, Sassolas G, and Rousset B. Identification of thyroid hormone residues on serum thyroglobulin: a clue to the source of circulating thyroglobulin in thyroid diseases. Eur J Endocrinol 140: 457–467, 1999.[Abstract]
11. Herzog V, Berndorfer U, and Saber Y. Isolation of insoluble secretory product from bovine thyroid: extracellular storage of thyroglobulin in covalently cross-linked form. J Cell Biol 118: 1071–1083, 1992.
12. Jung FF, Bachinsky DR, Tang SS, Zheng G, Diamant D, Haveran L, McCluskey RT, and Ingelfinger JR. Immortalized rat proximal tubule cells produce membrane bound and soluble megalin. Kidney Int 53: 358–366, 1998.[CrossRef][ISI][Medline]
13. Lisi S, Chiovato L, Pinchera A, Marcocci C, Menconi F, Morabito E, Altea MA, McCluskey RT, and Marinò M. Impaired thyroglobulin (Tg) secretion by FRTL-5 cells transfected with soluble receptor associated protein (RAP): evidence for a role of RAP in the Tg biosynthetic pathway. J Endocrinol Invest 26: 1105–1110, 2003.[ISI][Medline]
14. Lisi S, Pinchera A, McCluskey RT, Willnow TE, Refetoff S, Marcocci C, Vitti P, Menconi F, Grasso L, Luchetti F, Collins AB, and Marinò M. Preferential megalin-mediated transcytosis of low-hormonogenic thyroglobulin: a control mechanism for thyroid hormone release. Proc Natl Acad Sci USA 100: 14858–14863, 2003.
15. Lisi S, Segnani C, Mattii L, Botta R, Marcocci C, Dolfi A, McCluskey RT, Pinchera A, Bernardini N, and Marinò M. Thyroid dysfunction in megalin deficient mice. Mol Cell Endocrinol 236: 43–47, 2005.[CrossRef][ISI][Medline]
16. Marinò M, Chiovato L, Lisi S, Pinchera A, and McCluskey RT. Binding of the low density lipoprotein receptor-associated protein (RAP) to thyroglobulin (Tg): putative role of RAP in the Tg secretory pathway. Mol Endocrinol 15: 1829–1837, 2001.
17. Marinò M, Zheng G, Chiovato L, Pinchera A, Brown D, Andrews D, and McCluskey RT. Role of megalin (gp330) in transcytosis of thyroglobulin by thyroid cells: a novel function in the control of thyroid hormone release. J Biol Chem 275: 7125–7138, 2000.
18. Moreno JC, de Vijlder JJM, Vulsma T, and Ris-Stalpers C. Genetic basis of hypothyroidism: recent advances, gaps and strategies for future research. Trends Endocrinol Metab 14: 318–326, 2003.[CrossRef][ISI][Medline]
19. Norden AGW, Lapsley M, Igarashi T, Kelleher CL, Lee PJ, Matsuyama T, Scheinman SJ, Shiraga H, Sundin DP, Thakker RV, Unwin RJ, Verroust P, and Moestrup SK. Urinary megalin deficiency implicates abnormal tubular endocytic function in Fanconi syndrome. J Am Soc Nephrol 13: 125–133, 2002.
20. Orlando RA and Farquhar MG. Identification of a cell line that expresses a cell surface and a soluble form of the gp330/receptor-associated protein (RAP) Heymann nephritis antigenic complex. Proc Natl Acad Sci USA 90: 4082–4086, 1993.
21. Pohlenz J, Maqueem A, Cua K, Weiss RE, Van Sande J, and Refetoff S. Improved radioimmunoassay for measurement of mouse thyrotropin in serum: strain differences in thyrotropin concentration and thyrotroph sensitivity to thyroid hormone. Thyroid 9: 1265–1271, 1999.[ISI][Medline]
22. Schneider PB. Thyroidal iodine heterogeneity: "last come first served" system of iodine turnover. Endocrinology 74: 973–980, 1964.[ISI][Medline]
23. Willnow TE. Receptor-associated protein (RAP): a specialized chaperone for endocytic receptors. Biol Chem 379: 1025–1031, 1998.[ISI][Medline]
24. Willnow TE, Armstrong SA, Hammer RE, and Herz J. Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivo. Proc Natl Acad Sci USA 92: 4537–4541, 1995.
25. Willnow TE, Rohlmann A, Horton J, Otani H, Braun JR, Hammer RE, and Herz J. RAP, a specialized chaperone, prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors. EMBO J 15: 2632–2639, 1996.[ISI][Medline]
26. Zheng G, Bachinsky DR, Stamenkovic I, Strickland DK, Brown D, Andres G, and McCluskey RT. Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, gp330 and LRP/a2MR, and the receptor-associated protein (RAP). J Histochem Cytochem 42: 531–542, 1994.[Abstract]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
