Division of Endocrinology and Metabolism, 1 Department of Medicine, and the Departments of 2 Cell Biology and 3 Pathology, University of Alabama at Birmingham, and the 4 Veterans Affairs Medical Center, Birmingham, Alabama 35294
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ABSTRACT |
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In this study, we examined human growth hormone (hGH)-induced changes in nonionic detergent solubility characteristics of its receptor (hGHR). Exposure of IM-9 cells to hGH caused a time- and concentration-dependent loss of immunoblottable detergent-extractable hGHRs and a corresponding accumulation of receptors in a detergent-insoluble pool. At 37°C, the loss of detergent-soluble and the accumulation of detergent-insoluble hGHRs both preceded hGH-induced loss of total cell hGHRs. The detergent-insoluble receptor pool was progressively enriched in an apparent disulfide-linked form of the hGHR. Exposure to hGH at 4°C allowed hGH-induced hGHR disulfide linkage but did not promote changes in receptor detergent solubility, indicating that hGHR detergent insolubility cannot be explained solely by the formation of that linkage. Experiments carried out with hGH at 20°C and with the phorbol ester, phorbol-12,13-myristate acetate, at 37°C indicated that loss of detergent-soluble hGHRs can be uncoupled from accumulation of detergent-insoluble receptors. From these data, we envision at least two related, but separable, trafficking pathways taken by hGHRs after their surface interaction with hGH: 1) ligand-mediated endocytosis and degradation (accounting for only some of the receptors lost from the detergent-soluble fraction) and 2) ligand-mediated accumulation in a detergent-insoluble subcellular fraction (arising largely from receptors redistributed from the detergent-soluble fraction).
downregulation; receptor trafficking; protein sorting; signaling
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INTRODUCTION |
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BINDING OF A POLYPEPTIDE ligand to its cell surface receptor typically sets into motion a series of ligand-specific events. These include signal initiation, ligand-receptor internalization, signal desensitization, and intracellular routing and disposition (degradation and/or recycling) of ligand and receptor (28). Interaction of growth hormone (GH), a 22-kDa peptide hormone, with its receptor (GHR) leads to rapid dimerization of GHRs with formation of a 1:2 GH-GHR complex; this dimerization is believed obligate for GH signal transduction (7, 16). GH-induced GHR dimerization acutely leads to activation and tyrosine autophosphorylation of the GHR-associated cytoplasmic tyrosine kinase, JAK2, which promotes transient tyrosine phosphorylation of the GHR (2). These events initiate signal cascades that involve enzymatic activation of ras, mitogen-activated protein kinase, and phosphatidylinositol-3' kinase, as well as the recruitment of the latent cytoplasmic transcription factors, STATs 1, 3, and 5, to the GHR-JAK2 complex, where they are tyrosine phosphorylated and activated (2, 30). Shortly after initiation of this signaling, surface GHRs undergo internalization, a process dependent on the presence of a phenylalanine residue in the proximal one-third of the receptor cytoplasmic domain (1) and an intact cellular ubiquitination system (25). Although events that desensitize GH-induced signaling are incompletely understood, it is presumed that GHR internalization as well as GH-induced activation of protein tyrosine phosphatase(s) (12) contributes to this desensitization.
Multiple observations have been made regarding the fate of bound GH after the processes referred to above are initiated. Bound GH has been shown to be 1) internalized and degraded in lysosomes (4, 14), 2) exocytosed, either intact or degraded, from the cell (15), or 3) targeted to the nucleus, where its associations and functions are as yet unclear (20). The fate of GH-activated GHRs is even less clear, owing in large part to the paucity of direct-reacting reagents for use in tracking the receptor. Downregulation of GHRs (14, 18), defined as a decrease in cell surface GHR concentration after chronic prior exposure to GH, is presumed related to GHR internalization (1) and perhaps receptor degradation (24) or sequestration, but these phenomena have not been elucidated. Transient GH-induced association of the GHR with the nucleus has been observed and may have either a direct role in signaling or serve in this respect merely to transport GH to this subcellular destination (21).
In our previous studies, we have detected in human (h) IM-9 lymphoblasts the hGH-induced appearance of a high-Mr form of the hGHR when detergent-soluble cellular proteins are electrophoretically resolved under nonreduced conditions and anti-hGHR immunoblotted (10). Roughly one-half of the hGHRs that become acutely tyrosine phosphorylated in response to hGH treatment undergo this apparent disulfide linkage, although the role played by disulfide-linked hGHRs with regard to signaling and/or receptor trafficking is unclear. In this study we further examined the fate of activated receptors in IM-9 cells by monitoring the detergent extractability of hGHRs after stimulation of cells with hGH. We observed a quantitatively significant and progressive hGH-induced insolubility of hGHRs in Triton X-100 that cannot be accounted for exclusively by formation of the apparent disulfide-linked form of the receptor. Our results indicate the existence of a hGH-induced pool of long-lived hGHRs not previously detected biochemically in these cells.
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METHODS |
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Materials. Recombinant hGH was kindly provided by Eli Lilly (Indianapolis, IN). Phorbol-12,13-myristate acetate (PMA), staurosporine, cytochalasin B, nocodazole, and nystatin were purchased from Sigma Chemical (St. Louis, MO), as were routine reagents, unless otherwise noted.
IM-9 cell culture, stimulation, and protein extraction. IM-9 cells were maintained as described previously and stimulated with hGH or PMA after serum starvation for 16-20 h (10). Serum starvation was accomplished by substituting 0.5% (wt/vol) bovine serum albumin (BSA; fraction V, Boehringer Mannheim, Indianapolis, IN) for fetal calf serum in the culture medium. Serum-starved cells were resuspended at 25-50 × 106 cells/ml in binding buffer [BB; consisting of 25 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.1% (wt/vol) BSA, and 1 mM dextrose]. After a 15-min preincubation at the specified temperature for equilibration, hGH (added at a 1:40 dilution), PMA (added at a 1:100 dilution), or vehicle (BB or dimethyl sulfoxide, depending on the stimulus being controlled) was added for the indicated durations. For time course experiments, hGH, PMA, or BB was added such that all stimulations ended simultaneously.
Stimulations were terminated and cells were collected by centrifugation (800 g for 1 min at 4°C) and aspiration of the BB. Cell pellets were either processed directly or snap-frozen in liquid nitrogen and stored atElectrophoresis and immunoblotting. Resolution of proteins in the detergent-soluble, detergent-insoluble, and total cell extracts (prepared as above) under reduced or nonreduced conditions by SDS-polyacrylamide gel electrophoresis (PAGE), Western transfer of proteins, and blocking of nitrocellulose membranes [Hybond-enhanced chemiluminescence (ECL); Amersham] with 2% BSA were performed as previously described (10, 30). Anti-hGHR serums (directed at either the residue 317-620 or 271-620 regions of the cytoplasmic domain) were generated and affinity purified and used for immunoblotting, as has been previously described (10). Horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:1,500) and ECL detection reagents (all from Amersham) were used for detection of specifically recognized hGHRs, according to the manufacturer's suggestions.
Data analysis. Densitometry of ECL immunoblots was performed using a solid-state video camera (Sony-77, Sony) and a 28-mm MicroNikkor lens over a lightbox of variable intensity (Northern Light Precision 890, Imaging Research, Toronto, Canada). Quantification was performed using a Macintosh II-based image analysis program (Image 1.49, developed by W. S. Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MD). The fraction of hGHRs remaining in extracts (detergent-soluble or total) from hGH-treated cells was estimated by measuring by densitometry the intensity of the specifically detected hGHR signal relative to that signal present within the same experiment in extracts from unstimulated cells. As indicated when graphically shown, pooled data from numerous experiments are displayed as the means ± SE. The significances of differences of pooled results are estimated by unpaired t-tests.
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RESULTS |
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hGH induces detergent insolubility of the IM-9 hGH receptor. To examine the influence of hGH on the cellular level of hGHRs, IM-9 cells were exposed to the hormone at 37°C and evaluated by specific anti-hGHR immunoblotting. When cells were lysed in a buffer containing 0.2% Triton X-100, the level of immunoblottable hGHRs in the detergent-soluble cell extract was observed to progressively decline after exposure to hGH for 30 min or longer (Fig. 1A). To determine whether this hGH-induced loss of detergent-soluble hGHRs reflected a loss of total cell hGHRs, cells were solubilized directly in SDS (1%) sample buffer, and the resulting total cell extract was analyzed by anti-hGHR immunoblotting. Very little change in the level of total hGHRs was detected after even 120 min of hGH exposure (Fig. 1B).
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The pool of hGH-induced detergent-insoluble hGHRs includes disulfide-linked hGHRs. We investigated the distribution of the hGH-induced apparent disulfide-linked hGHR form in detergent-soluble and -insoluble fractions (Fig. 3). As previously observed (10), the level of detergent-soluble disulfide-linked hGHRs declined after 60 min of hGH exposure. In contrast, detergent-insoluble hGHRs progressively accumulated as the duration of hGH treatment increased to 90 min. Notably, the accumulation of the disulfide-linked hGHR form in the detergent-insoluble fraction was more progressive than that of the non-disulfide-linked hGHR. Thus the disulfide-linked hGHR, a form associated with hGHR activation (10), comprises a significant component of the detergent-insoluble hGHRs that appear in response to hGH. Interestingly, as we found for hGH-induced disulfide-linked hGHR formation (10), tyrosine kinase activation was apparently not required for hGH-induced changes in hGHR detergent solubility. Pretreatment with staurosporine [1.25 µM, a concentration that inhibits detectable hGH-induced tyrosine phosphorylation in IM-9 cells (10)] did not affect hGH-induced loss of detergent-soluble hGHRs or their accumulation in the detergent-insoluble fraction (not shown).
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hGH-induced accumulation of detergent-insoluble hGHRs is temperature sensitive. To address whether hGH-induced detergent insolubility of hGHRs and hGH-induced formation of apparent disulfide-linked hGHRs were related phenomena, we considered whether disulfide linkage might be sufficient to confer detergent insolubility to the hGHR. To approach this, the abilities of hGH to promote hGHR disulfide linkage and alterations in hGHR detergent solubility were examined at low temperatures.
As shown in Fig. 4A, stimulation of IM-9 cells at 4°C, a temperature that prevents receptor trafficking other than that associated with lateral movement within the membrane, allowed continued detectability of the hGH-induced disulfide-linked detergent-soluble hGHR form. Formation of disulfide-linked hGHRs was somewhat slower at 4°C than at 37°C but was nonetheless easily detectable at this lower temperature. In contrast, no hGH-induced loss of detergent-soluble hGHRs (Fig. 4B) or accumulation of detergent-insoluble hGHRs [either under reduced conditions, as in Fig. 4C, or under nonreduced conditions (data not shown)] was noted after as long as 120 or 240 min, respectively, of hGH exposure. This inability to detect changes in hGHR detergent solubility under conditions that allow hGH-induced formation of disulfide-linked hGHRs indicates that hGH-induced accumulation of detergent-insoluble hGHRs cannot be explained solely by diminished detergent solubility of the disulfide-linked form.
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Phorbol ester treatment induces loss of detergent-soluble hGHRs by a mechanism apparently distinct from that of hGH. Both hGH and phorbol ester treatments of IM-9 cells are known to cause downregulation of hGHRs (18, 26). In IM-9 cells, roughly one-half of surface hGHRs are lost by treatment of cells at 37°C with 440 ng/ml hGH for 45 min (18) or with the phorbol ester PMA (greater than or equal to 1 µM) for 30 min (26). The mechanisms of these downregulations are unclear, but the hGH-induced downregulation is believed to be related more to acceleration of disposition of hGHRs than to diminution in their synthesis (18).
Given their similarity in decreasing IM-9 cell surface hGHR levels, we examined whether hGH and PMA also exerted similar effects on hGHR detergent solubility. Both PMA and hGH induced a loss of detergent-soluble hGHRs (Fig. 6A). As shown earlier, the time course of the hGH-induced effect was characterized by significant and progressive loss of soluble hGHRs after 30 min. The PMA-induced loss of detergent-soluble hGHRs was quantitatively similar to that caused by hGH but was nearly maximal by 30 min (Fig. 6, A and B). In contrast to hGH, PMA treatment induced no accumulation of full-length hGHRs in the detergent-insoluble fraction. Instead, the low level of hGHRs present in this fraction basally was further reduced by PMA in a fashion parallel to the effect of PMA on detergent-soluble hGHRs (Fig. 6B). In other experiments, the acute loss of total cell full-length hGHRs in response to PMA was further demonstrated by direct solubilization of the cells in hot SDS sample buffer before electrophoresis and immunoblotting (J. Alele, J. F. Goldsmith, J. Jiang, and S. J. Frank, unpublished observations).
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DISCUSSION |
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By anti-hGHR immunoblotting, we observe in the IM-9 B-lymphoblastoid cell line that hGH causes time- and concentration-dependent loss of detergent-soluble hGHRs and accumulation of detergent-insoluble hGHRs. The time courses of the two changes are roughly parallel, with the loss of detergent-soluble receptors likely slightly preceding their appearance in the detergent-insoluble fraction. Both changes are progressive and most readily detectable after 30 min of hGH stimulation, and both precede the hGH-induced loss of SDS-extractable total cell immunoblottable hGHRs. We find that the detergent-insoluble pool of hGHRs that arises in response to hGH is markedly and progressively populated with the apparent disulfide-linked hGHR form. Because this form is detectable as early as 1 min into stimulation and is acutely tyrosine phosphorylated (10), we conclude that a significant fraction of detergent-insoluble receptors arises from a pool of activated hGHRs. Thus our findings indicate that, in IM-9 cells at 37°C, it is likely that hGH-induced disposition of detergent-soluble hGHRs is not entirely via ligand-induced receptor degradation but rather is also contributed to by redistribution of previously activated receptors to a detergent-insoluble subcellular fraction. We note that if hGH stimulation is performed at 20°C rather than at 37°C, there is also loss of detergent-soluble hGHR (Fig. 5A); however, there is no accompanying accumulation of detergent-insoluble hGHRs (Fig. 5B). Thus the phenomena of hGH-induced loss of detergent-soluble hGHRs and accumulation of detergent-insoluble hGHRs can be uncoupled at 20°C.
Furthermore, treatment of cells with PMA at 37°C promotes rapid and dramatic loss of detergent-soluble hGHRs that is also not accompanied by accumulation of detergent-insoluble hGHRs. However, this loss of detergent-soluble receptors appears to be mechanistically different from that promoted by hGH. Although we are as yet uncertain as to their exact identification, the size and immunoreactivity of the 65- to 68-kDa bands (Fig. 6, A and B), the detection of which is increased in response to PMA but not in response to hGH, are consistent with their being "remnants" (fragments that include the transmembrane and cytoplasmic domains) generated after selective proteolysis of mature GHRs at a site(s) at or near the membrane. Such selective proteolysis in response to PMA has been observed for other membrane proteins, the extracellular domains of which are "shed" as water-soluble proteins (3). We speculate that in IM-9 cells PMA causes loss of detergent-soluble hGHRs (and likely hGHR downregulation) in part by inducing shedding of the extracellular domain of the membrane hGHR [thereby generating the water-soluble hGH binding protein (5, 13, 27)] without redistributing hGHRs to another (i.e., detergent-insoluble) subcellular fraction. In contrast, it is clear that in IM-9 cells hGH-induced loss of detergent-soluble receptors appears not to be accompanied by such a selective hGHR proteolysis.
We note the recent findings of King et al. (17) in which PMA-induced acute loss of cell surface GHRs in murine 3T3-F442A fibroblasts was apparently not accompanied by loss of total cell GHRs and was therefore attributed to an intracellular redistribution of receptors. We cannot yet explain the differences between our findings and those of King et al. on this matter, but we note the marked differences between species in mechanisms of generation of the GH binding protein (5). Given that rodents are not thought to generate the GH binding protein by cleavage of the transmembrane GHR as much as by alternative splicing of GHR mRNA (5), we speculate that the sensitivity to PMA for the elicitation of such a cleavage might differ between rodent and human cell lines. We are currently testing this hypothesis.
Although the hGH-induced detergent-insoluble pool of hGHRs is well populated with the disulfide-linked hGHR, the stimulations performed at 4°C result in hGHR disulfide linkage without acquisition of detergent insolubility, indicating that disulfide linkage is not sufficient for hGH-induced hGHR detergent insolubility. Whether hGHR disulfide linkage, which occurs in response to hGH apparently at the cell surface, is required for disposition of hGHRs to a detergent-insoluble pool is as yet uncertain.
Detergent insolubility of receptors with potential relevance to signaling has been observed in other systems (6, 11). In these cases, detergent insolubility is thought to be related to association of the receptors with the cytoskeleton. Our studies in IM-9 cells have not yet conclusively indicated such an association for the hGHR in response to hGH. The mild diminution that we observed in the levels of both basal detergent-soluble hGHRs and hGH-induced detergent-insoluble hGHRs in the presence of cytochalasin B is consistent with previous data indicating that cytochalasins by themselves decrease cell surface hGHR expression in IM-9 cells by undefined mechanisms (29). These data, coupled with the lack of effect of nocodazole on the process, lead us to conclude that hGH-induced hGHR detergent insolubility in IM-9 cells is independent of the integrity of microtubules and is, at most, only partially dependent on an intact actin cytoskeleton.
Our biochemical observation of hGH-induced hGHR detergent insolubility in IM-9 cells raises two major questions that will be the focus of future studies: 1) Do detergent-insoluble hGHRs, which appear to in part derive from an activated pool of hGHRs, reside preferentially in a particular subcellular location or organelle? and 2) Does the acquisition of detergent insolubility allow this pool of hGHRs to perform a particular role, for example, in hGHR signaling or trafficking, in response to hGH?
In regard to the first question, GHRs have been observed in certain cell types to undergo translocation to the nucleus in a phenomenon thought to be related to the appearance of GH in that organelle (20, 21). We think it unlikely on several grounds that the hGH-induced accumulation of detergent-insoluble hGHRs that we observe reflects such a nuclear translocation. In previous radioautographic ultrastructural studies of the uptake of 125I-hGH by IM-9 cells, no nuclear localization of the labeled hormone was detected over a time course of 180 min of stimulation (4), leading us to believe that IM-9 cells may not behave similarly to other cell types studied in this respect. Second, when observed, GHR nuclear localization in Chinese hamster ovary cell-rat GHR transfectants was acute (within 10 min of GH treatment) and transient (redistributed to nonnuclear areas of the cell in a pattern identical to untreated cells by 60 min into GH exposure) (21). This is in contrast to our observed time course of hGH-induced hGHR detergent insolubility in IM-9 cells (most easily detectable after 30 min and progressive for at least 2 h). Finally, in our experiments, even when extraction was performed with up to 4% Triton X-100, hGHRs still became detergent insoluble in response to hGH. This concentration of Triton X-100 would be expected to solubilize nuclear membrane-associated GHRs (21).
Other potential subcellular localizations have been described that could, in principle, include detergent-insoluble GHRs. Caveolae, for example, are detergent-insoluble submembranous vesicles that have been noted to be enriched in signaling molecules such as growth factor receptors and other tyrosine kinases and substrates (19). Typically, treatment of cells with the antibiotic nystatin disrupts caveolae formation (23). Our preliminary experiments indicate that incubation of cells with nystatin (50 µg/ml) does not prevent the hGH-induced acquisition of hGHR detergent insolubility (data not shown), leading us to tentatively conclude that detergent-insoluble hGHRs are not associated with caveolae.
"Capping" of hGH (and therefore presumably of hGHRs as well) has been described in IM-9 cells (9). Whereas their detergent solubility characteristics are unknown, these hGH caps form within 30 min of stimulation at one pole of the cell; their function and disposition are also unknown. Studies of the capping of hormones other than GH and of other ligands by lymphocytes indicate that caps are intimately associated with the actin cytoskeleton, although they can be at least partially solublized in nonionic detergents (22). Morphological studies of hGHR distribution in response to hGH may allow us to discern whether capping of hGHRs correlates with hGHR detergent insolubility in our system.
Regarding the second question raised by our observations (Is there a particular function to be ascribed to the detergent-insoluble hGHR?), we note that the time course of its formation is similar to the time courses observed in IM-9 cells for the phenomena of 1) hGH-induced hGHR downregulation (14, 18), 2) the appearance of a nondissociable component of 125I-hGH binding (14), and 3) the exocytic release of previously internalized hGH from the cell (15). These three phenomena may be related. Notably, exocytosis of internalized hGH nearly ceased at 20°C, whereas endocytosis continued (15). This uncoupling may be related to the uncoupling of hGH-induced loss of detergent-soluble hGHRs and accumulation of detergent-insoluble hGHRs that we have observed at that temperature.
In summary, our data indicate that in IM-9 cells hGHRs undergo at least two related, but separable, intracellular trafficking routes in response to hGH. We envision that the loss of detergent-soluble hGHRs likely reflects in part their endocytosis and degradation, presumably similar to a pathway followed by many receptors. Additionally, another hGHR routing pathway in these cells that involves the accumulation of detergent-insoluble receptors is activated by hGH at 37°C. hGHRs routed to this pathway are long-lived and are enriched in the apparent disulfide-linked receptor form. We speculate that the second pathway, by serving either as a delivery route for hGH exocytosis or as a vehicle for receptor sequestration, may be involved in receptor downregulation and may therefore be important in modulation of hormonal responses. Furthermore, as a practical matter, our findings sound a cautionary note that the evaluation of the fate of ligand-activated receptors, in general, may not be complete without an analysis of those receptors that are rendered insoluble in the standard nonionic detergents often used for extraction.
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ACKNOWLEDGEMENTS |
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We appreciate helpful conversations with Drs. J. Kudlow, A. Paterson, E. Chin, L. Liang, S.-O. Kim, J. Alele, W. Bradley, J. Baker, J. Couchman, J. Collawn, P. Atkinson, and A. Wells.
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FOOTNOTES |
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The authors gratefully acknowledge Eli Lilly Co. for providing the hGH. During some of the period of these studies, S. J. Frank was an Early Career Development Award recipient from the American Federation for Clinical Research. This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46395 (to S. J. Frank), a Veterans Affairs Merit Review award (to S. J. Frank), and a Medical Scientist Training Program grant (to J. F. Goldsmith).
Address for reprint requests: S. J. Frank, Univ. of Alabama at Birmingham, Rm. 756, DREB, UAB Station, Birmingham, AL 35294.
Received 16 April 1997; accepted in final form 24 July 1997.
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