Multiple Internalization Motifs Differentially Used by Prolactin Receptor Isoforms Mediate Similar Endocytic Pathways
Juu-Chin Lu,
Patricia Scott1,
Ger J. Strous and
Linda A. Schuler
Endocrinology-Reproductive Physiology Program (J.-C.L., L.A.S.), Department of Comparative Biosciences (J.-C.L., P.S., L.A.S.), University of Wisconsin-Madison, Madison, Wisconsin 53706; and Department of Cell Biology (G.J.S.), University Medical Center Utrecht and Institute of Biomembranes, 3584 CX Utrecht, The Netherlands
Address all correspondence and requests for reprints to: Linda A. Schuler, Department of Comparative Biosciences, University of Wisconsin, 2015 Linden Drive, Madison, Wisconsin 53706. E-mail: schulerl{at}svm.vetmed.wisc.edu.
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ABSTRACT
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Prolactin (PRL) regulates a variety of physiological processes, including mammary gland growth and differentiation, modulation of behavior, and immune function. A long PRL receptor (lPRLR) and short (sPRLR) isoform were identified in ruminants and rodents, which differ in their distal cytoplasmic domains and possess markedly distinct signaling capacities. Here we compared endocytosis of the bovine isoforms and found that the lPRLR internalized faster than the sPRLR, which would contribute to short-term down-regulation of lPRLR signaling at targets expressing both isoforms. Multiple motifs were required to mediate internalization of the lPRLR, including a phenylalanine (F290) plus a nearby dileucine, and three dileucines proximal to amino acid 272. This is different from the closely related GH receptor that requires only the phenyl-alanine-containing motif for endocytosis. Truncated lPRLR (cT272), which is the same length as the sPRLR and contained the proximal three dileucines, internalized at the same rate as the full-length lPRLR. Finally, the two dileucines shared by the sPRLR were able to mediate similar endocytic pathways as the lPRLR, as revealed by overexpression of mutant dynamin and clathrin hub, despite the slower rate. These studies define the basis of cellular trafficking of PRLR isoforms and increase our understanding of control of target cell responsiveness by PRL.
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INTRODUCTION
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THE PITUITARY HORMONE, prolactin (PRL) (1), and its placental relatives such as placental lactogen (PL) modulate the development and activity of a wide range of fetal and adult tissues, including multiple targets in the reproductive system such as the mammary gland, the liver, and the immune system (for reviews, see Refs. 1, 2, 3). The PRL receptor (PRLR) is a member of the cytokine receptor superfamily, which shares several features including multiple motifs in the extracellular domain important for ligand binding, a single transmembrane region, several conserved regions in the proximal cytoplasmic domain, and the lack of intrinsic kinase activity. Ligand binding to these receptors leads to dimerization (in the case of the PRLR) or oligomerization, which activates kinases, including Janus kinases (JAKs), thereby transmitting signals to appropriate cell compartments.
Alternative splicing gives rise to PRLR with distinct cytoplasmic domains (4, 5). In rodents and ruminants, the PRLR is expressed as so-called long (lPRLR) and short (sPRLR) isoforms. Although different mechanisms are involved in rodents and ruminants, all these sPRLRs diverge from the long isoform and from each other at the same point in the proximal cytoplasmic domain shortly after the conserved proline-rich region (box 1) equivalent to amino acid 261 of the rat sequence. These severely truncated receptors terminate in short unique C-terminal tails, for a total of 38 amino acids in the cytoplasmic domain in the bovine sPRLR (6, 7, 8). The ability of all short isoforms to transduce signal through characterized PRL signaling pathways is more limited than that of the lPRLR. The rat sPRLR has been shown to promote proliferation in some, but not all, studies (9, 10). However, it was unable to mediate transcriptional activation via the JAK-signal transducer and activator of transcription pathway (8, 10, 11, 12). Furthermore, sPRLR can inhibit lPRLR activation of JAK2 and transcription (9, 13) via formation of heterodimers. The possibility of distinct pathways mediated by the sPRLR has also been reported (14). All target tissues examined contain PRLR transcripts for both long and short isoforms, and levels vary with cell type and physiological status (8, 15). Humans also express multiple PRLR isoforms, including recently described isoforms with very limited cytoplasmic domains (16, 17, 18). However, considerably less is known about these forms. The differences in signaling capacity, along with coexpression and ability to heterodimerize resulting in lPRLR signal attenuation, indicate that the relative level of these PRLR isoforms is an important determinant of the cellular response to PRL.
Ligand binding to many membrane receptors also initiates internalization of both ligand and receptor. This process results in multiple potential fates, including recycling of receptors back to the surface, degradation of ligand and/or receptor by lysosomes or proteasomes resulting in down-regulation of receptor, and transport to other cellular compartments to mediate signaling. This process is therefore a major modulator of cell responsiveness over the short term. However, relatively little is known about internalization of the PRLR and related receptors. Studies of the GH receptor (GHR), which shares multiple regions of similarity with the PRLR, have demonstrated that it is internalized both by clathrin-coated pits (19), as well as via caveolae (20). Furthermore, several approaches have demonstrated that the GHR requires the ubiquitin-conjugating system for endocytosis (for review, see Ref. 21). Internalization motifs for the rat sPRLR have been suggested by deletion mutagenesis (22). However, nothing is known about the lPRLR, as well as other sPRLRs that contain unique cytoplasmic termini.
The distinct cytoplasmic sequence and signaling capabilities of the lPRLR and the sPRLR suggest different receptor processing in response to ligand. Here we have compared internalization of the long and short isoforms of the bovine PRLR and have found that the lPRLR is internalized more rapidly. In cells expressing both isoforms of the receptor, differences in ligand-induced internalization would lead to changes in relative amounts of each receptor expressed at the cell surface and, therefore, changes in signals transduced. To understand the mechanism of internalization of PRLR isoforms, we delineated cytoplasmic residues involved in regulating internalization. Using deletions and point mutations, we have identified two regions required for internalization of the lPRLR: one containing a Phe residue and a nearby dileucine sequence, and the other containing three dileucine sequences proximal to amino acid 272. The first region is unique to the lPRLR and may provide differential processing of the isoforms, leading to regulation of PRL responsiveness. The first two dileucines in the second region are also conserved in the sPRLR, suggesting their role in internalization of this isoform.
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RESULTS
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Internalization of the Long and Short Isoforms of the PRLR
The PRLR and GHR belong to the cytokine receptor superfamily. These receptors are single transmembrane proteins composed of a ligand-binding extracellular domain, a transmembrane domain, and a cytoplasmic domain (Fig. 1
). The sPRLR and lPRLR isoforms result from alternative RNA splicing and differ only in their distal cytoplasmic domains. PRLRs are highly conserved across species and also share several structural features and sequences with the closely related GHR, including those implicated in internalization, as shown.

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Figure 1. Diagrammatic Representation of the PRLR/GHR Forms
The PRLR and the GHR contain an extracellular domain (ECD), a transmembrane domain (TM), and a cytoplasmic domain (CYD). In rodents and ruminants, long and short PRLR isoforms are generated by alternative splicing. The hatched bars indicate C-terminal tails of the sPRLR isoforms, which are unique to the short forms and differ among rodents and ruminants. The phenylalanine residue, previously identified in the UbE motif of the rabbit GHR at position 327 (26 ), is marked (F). Phenylalanine residues in a similar context are found in other GHRs and lPRLRs in virtually all species studied except for primates, birds, and fish PRLRs, which contain a tyrosine (Y) in that position. LL or IL denotes the positions of the dileucine sequences of the bovine PRLRs examined herein and the corresponding dileucine sequences in the PRLRs and GHRs. The LL denotes the dileucine motif unique to the GHR that can play a role in internalization under some circumstances (52 ).
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Internalization of the PRLR isoforms was compared in COS-7 cells transiently transfected with lPRLR or sPRLR by following 125I-labeled ligand bound to surface receptors under conditions that prevented internalization, and then allowing internalization to proceed by incubation at 37 C. After various times, surface and internalized ligand associated with the cells and intact and degraded ligand in the media were determined. Internalization of 125I-bPRL and 125I-bPL did not differ for either isoform (data not shown). As shown in Fig. 2
, the percentage of internalized ligand relative to total binding at each time point was greater for the lPRLR than sPRLR (P < 0.05 at times of 15 min and later). The rate of internalization, calculated for 030 min (before appreciable degradation), was also greater for the lPRLR (slope lPRLR = 0.01847%/min; slope sPRLR = 0.01225%/min).

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Figure 2. Internalization Kinetics of Bovine Long and Short PRLR Isoforms
Ligand internalization in COS-7 cells transiently expressing the lPRLR or sPRLR was determined, and internalization ratio of the lPRLR and sPRLR at different times after initiation of internalization was calculated as described in Materials and Methods. Each point represents the mean ± SE of triplicate measurements from at least three independent experiments. When not shown, SE bars are smaller than the symbol.
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Degraded ligand released into the media after incubation at 37 C was examined by determining trichloroacetic acid-soluble radioactivity. No appreciable degradation was detected in the media through 15 min, consistent with previous reports (23). However, trichloroacetic acid-soluble radioactivity steadily accumulated thereafter (after 2 h: 58.8 ± 15.7% of initial binding for the lPRLR and 29.5 ± 8.4% for the sPRLR, respectively).
Differences in the cytoplasmic domains of the receptors may alter not only interactions with endocytic pathways, but also trafficking within the cell, both of which may alter cell surface expression. Changes in surface levels may alter observed rates of internalization not only by affecting ligand binding, but also by competition for endocytic machinery. Therefore, we verified that our assay was independent of surface expression levels over the ranges examined by modulating expression of each of the PRLR isoforms over a range greater than 6-fold. The internalization ratios at both 15 and 30 min for each isoform did not differ with expression (Table 1
), nor was there a correlation of internalization ratio with surface expression (lPRLR: P = 0.441; sPRLR: P = 0.293). The expression levels of the different PRLR mutants examined in these studies were well within this range.
Identification of Internalization Motifs in the Truncated lPRLR and sPRLR Isoforms
The difference in the rate of internalization of the two receptor isoforms suggests that internalization motif(s) may be located in the unique portion of the cytoplasmic domain of the lPRLR, or that the 11-amino-acid unique C terminus of the sPRLR may play a negative role in receptor internalization. To identify motif(s) required for lPRLR internalization, we generated C-terminal truncated mutants of the lPRLR. Deletion to amino acids 453 (cT453), 415 (cT415), and 368 (cT368) did not significantly alter internalization (Fig. 3A
), suggesting that no important motifs were located in these regions or that positive and negative elements neutralized one another. Further truncation (cT311) internalized somewhat faster than cT368 at 15 min (P < 0.01), but was indistinguishable at 30 min, suggesting a possible negative element(s) located between amino acids 311 and 368. Deletion to amino acid 272 (cT272), yielding a PRLR the same length as the sPRLR although differing in the final 11 amino acids, internalized similarly to the wild-type lPRLR but slower than cT311, implying endocytosis motif(s) located between amino acids 272 and 311.

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Figure 3. Internalization of Wild-Type and C-Terminal Deletion Mutant lPRLRs
A and B, Ligand internalization in COS-7 cells transiently expressing the wild-type lPRLR, sPRLR, or mutant lPRLRs was determined as described in Materials and Methods. Each point represents the mean ± SE of triplicate measurements from at least three independent experiments. Schematic representation of the transmembrane and cytoplasmic domain of the wild-type and mutant receptors is shown in the right panel. When not shown, SE bars are smaller than the symbol. For A, different letters denote significant differences at 30 min (P < 0.05). The asterisk denotes the significant difference of cT311 at 15 min from the others. For B, different letters denote significant differences at both 15 and 30 min (P < 0.05).
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Since deletion of the cytoplasmic domain to amino acid 272 (cT272) resulted in similar internalization as the wild-type lPRLR, this suggested that other internalization motif(s) may be located proximal to amino acid 272.
As shown in Fig. 3B
, additional deletion of the lPRLR from amino acid 272 to 261 (cT261), reducing the receptor to sequence shared between the two isoforms, decreased receptor internalization compared with cT272, suggesting internalization motif(s) in this region of the lPRLR. Internalization of cT261 was not significantly different from the sPRLR, indicating that the unique tail of the sPRLR does not appear to contain either positive or negative elements. Failure of this unique C terminus of the sPRLR to alter receptor internalization when inserted into corresponding position of the lPRLR also supports this conclusion (data not shown). Deletion to amino acid 241 (cT241) further reduced receptor internalization to the same level as a mutant truncated immediately after the transmembrane domain (cT234), implying that common internalization motif(s) shared by both isoforms are located between amino acids 241 and 261.
Examining the lPRLR between amino acids 241 and 272, we found three dileucine motifs (IL243/244, LL259/260, LL268/269), two in the common region, and the final one unique to the lPRLR (Fig. 4A
). Many studies have demonstrated the importance of these dipeptides as internalization motifs and/or lysosomal targeting signals in membrane receptors (24, 25). Mutation of one individual dileucine pair to two alanines in the truncated lPRLR cT272 (cT272-IL243/244AA, LL259/260AA, LL268/269AA) reduced internalization compared with cT272 (Fig. 4
, B and C). Simultaneously mutating two dileucine pairs (cT2724L-A, in which LL259/260 and LL268/269 were mutated to alanines) reduced internalization slightly more. However, mutation of all three dileucine sequences (cT2726L-A) reduced internalization to the rate of cT234 (Fig. 4
, B and C), suggesting that together these three dileucine motifs play cooperative roles in the internalization of the truncated mutant cT272. Since the first two dileucine sequences (IL243/244, LL259/260) are located in the cytoplasmic region shared with the sPRLR, it is likely that the sPRLR uses these two dileucine sequences for internalization.

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Figure 4. Three Dileucine Sequences Proximal to Amino Acid 272 Mediate Internalization of the Truncated lPRLR cT272
A, The amino acid sequences of the proximal cytoplasmic domain of the lPRLR and sPRLR are shown. The dileucine motifs in this region (three in the lPRLR and two in the sPRLR) are underlined. The conserved box 1 region is enclosed by a rectangle. The positions of deletion mutants examined in Fig. 3B are indicated. B, Internalization of truncated lPRLR-cT272 and dileucine-mutated receptors. Ligand internalization in COS-7 cells transiently expressing the wild-type lPRLR, sPRLR, or mutant lPRLRs was determined as described in Materials and Methods. Each point represents the mean ± SE of triplicate measurements from at least three independent experiments. When not shown, SE bars are smaller than the symbol. Schematic representation of the transmembrane and cytoplasmic domain of the wild-type and mutant receptors is shown in the right panel. The positions of dileucine point mutations are indicated (x). C, Statistical comparisons of the data. NS, Not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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To determine the role of these dileucine sequences in internalization of the full-length lPRLR, point mutants of individual or double (4L-A) dileucine pairs were generated in the native lPRLR. In this context, they failed to alter internalization, even when two dileucine pairs were mutated simultaneously (Fig. 5
). However, when all three dileucines were simultaneously mutated (6L-A), the rate of internalization dropped significantly, although not to the level of the mutant receptor lacking a cytoplasmic domain. These data suggest that these dileucine sequences are necessary, but not sufficient, for optimal internalization of the full-length lPRLR.

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Figure 5. The Dileucine Sequences Proximal to Amino Acid 272 Have Less Effect on Internalization of the Full-Length lPRLR
Ligand internalization in COS-7 cells transiently expressing the wild-type or mutant lPRLRs was determined as described in Materials and Methods. Each point represents the mean ± SE of triplicate measurements from at least three independent experiments. When not shown, SE bars are smaller than the symbol. Schematic representation of the transmembrane and cytoplasmic domain of the wild-type and mutant receptors is shown on the right panel. The positions of three proximal dileucines in the wild-type lPRLR are indicated, as are point mutations (x). Different letters denote significant differences (P < 0.05).
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Identification of Internalization Motifs in the Full-Length lPRLR
To identify internalization motif(s) responsible for internalization of the full-length lPRLR, we examined the sequence between amino acids 272 and 311, since cT311 internalized faster than cT272 (Fig. 3A
). This region contains a phenylalanine residue (F290) in a context similar to the phenylalanine identified in the ubiquitin-dependent endocytosis (UbE) motif of the GH receptor (EFIEVD in the lPRLR and EFIELD in the GHR (Fig. 1
and Ref. 26). Mutation of this single phenylalanine to alanine has been shown to completely abolish internalization and ubiquitination of the GHR (19, 27). Therefore, the point mutant lPRLR F290A was generated to determine whether it played a similar role in internalization of the lPRLR.
lPRLR F290A internalized somewhat more slowly than the wild-type lPRLR (20% less, Fig. 6A
). However, this rate was significantly more rapid than that of the sPRLR. In contrast, the analogous mutant rabbit GHR construct was internalized dramatically more slowly than the wild-type GHR (60%) in a parallel assay as previously reported (Fig. 6A
and Ref. 19). These data indicated that although the F290 in the lPRLR appears to participate in regulation of internalization, it does not seem to be as important for the lPRLR as for the closely related GHR. Furthermore, the observed rates of internalization of the lPRLR and wide-type GHR were quite different. After 15 min, about twice as much lPRLR had been internalized compared with the GHR. These data suggest different mechanisms of endocytosis for these receptors.

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Figure 6. A Phenylalanine Residue (F290) and a Nearby Dileucine (LL286/287), in Combination with Three Proximal Dileucine Sequences, Mediate Internalization of the Wild-Type lPRLR
A, Ligand internalization in COS-7 cells transiently expressing the lPRLR, sPRLR, or lPRLR F290A was determined as described in Materials and Methods (left). For comparison, ligand internalization of the rabbit GHR or GHR F327A was also determined (right). B, Ligand internalization in COS-7 cells transiently expressing wild-type or point mutant PRLRs. Each point represents the mean ± SE of triplicate measurements from at least three independent experiments. When not shown, SE bars are smaller than the symbol. Schematic representation of the transmembrane and cytoplasmic domain of the wild-type and mutant receptors is shown in the right panel. The positions of dileucines and phenylalanine (F290) in the wild-type lPRLR are indicated, as are point mutations (x). Different letters denote significant differences (P < 0.05).
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We observed an additional dileucine sequence (LL286/287) located close to F290, which is conserved in virtually all of the long forms of the PRLRs examined, but in none of the GHRs. Mutation of this dileucine (LL286/287AA) decreased lPRLR internalization to the level of the F290A (Fig. 6B
). Simultaneously mutating F290 and dileucine LL286/287 (F290A LL286/287AA) further reduced receptor internalization to the level of the sPRLR, suggesting that both contributed to internalization of full-length lPRLR.
Because the point mutant F290A LL286/7AA still internalized more rapidly than the cT234 mutant without the cytoplasmic domain, although only at the rate of the sPRLR, we examined the role of the dileucine motifs proximal to amino acid 272 in combination with the region containing F290 and LL286/287. As shown in Fig. 6B
, mutating two of the proximal dileucines in addition to F290A (F290A 4L-A) further reduced internalization to the level of F290A LL286/7AA, compared with F290A alone. This result contrasted with the lack of effect observed when the same two proximal dileucines were mutated in the context of wild-type lPRLR (4L-A, Fig. 5
). When these two proximal dileucines were mutated in combination with both F290A and LL286/7AA (F290A LL286/7AA 4L-A), internalization was further reduced to the level of cT234, which contains no cytoplasmic domain. These data demonstrated that F290, LL286/287, and the proximal dileucines are all necessary for optimal internalization of the full-length lPRLR.
Endocytic Pathway(s) for PRLR Isoforms
The internalization pathways used by membrane receptors have implications for receptor and ligand processing as well as signaling. The involvement of the clathrin-mediated endocytic pathway and the caveolar pathway in internalization of PRLR isoforms was examined by inhibiting the function of dynamin, a GTP-binding protein shown to be a key component in both pathways (28, 29, 30). Disruption of the GTP binding site of dynamin (K44E) results in dominant inhibition of endogenous dynamin function (28). A truncated bovine clathrin heavy chain (T7-Hub), shown to be a dominant-negative inhibitor of clathrin vesicle formation (31), was also used to examine the involvement of the clathrin-mediated pathway in PRLR internalization.
As shown in Fig. 7
, overexpression of K44E-dynamin-1, but not wild-type dynamin-1, markedly inhibited both lPRLR and sPRLR internalization to the level of the mutant cT234 (compare with Fig. 3B
). Overexpression of T7-Hub also inhibited PRLR internalization, but not to the extent of K44E-dynamin-1. These data suggest that the clathrin-mediated pathway and/or the caveolar pathway may be involved in internalization of both the lPRLR and the sPRLR, and that signal(s) required for dynamin-mediated internalization of the PRLRs may be located in the common regions of the PRLR cytoplasmic domains.

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Figure 7. PRLR Internalization Is Inhibited by Mutant Dynamin and Clathrin Hub
Wild-type dynamin-1, K44E-dynamin-1, or T7-Hub was transiently cotransfected with lPRLR or sPRLR, into COS-7 cells, and ligand internalization was determined as described in Materials and Methods. Each point represents the mean ± SE of triplicate measurements from at least three independent experiments. When not shown, SE bars are smaller than the symbol. Asterisks denote significant differences from internalization of receptor+pcDNA3 vector (P < 0.05).
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DISCUSSION
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Cell responsiveness is regulated, in part, by surface expression of receptors. Ligand-induced internalization can lead to multiple potential fates of ligand and the receptor, including degradation of the receptor by lysosomes or proteasomes, which results in down-regulation by reducing the levels of surface receptor available for further signaling. In cells expressing different receptor isoforms that possess different signaling capacities or pathways, differential internalization of receptor isoforms in response to ligand can change the ratio of isoforms resulting in altered cellular responsiveness (32). The present studies have shown that the lPRLR internalizes faster than the sPRLR. Since target cells express both isoforms (8, 15), ligand exposure would lead to a relative increase in surface sPRLR, further decreasing signaling through the remaining lPRLR, by increasing formation of heterodimers.
Using deletions and point mutations, we identified two regions containing internalization motifs used by the lPRLR, as shown in Fig. 8
. The first region, containing a Phe residue (F290) and a nearby dileucine (LL286/287), is unique to the lPRLR, and both elements are necessary for optimal endocytosis. A second more proximal region contains three dileucines and is also required for optimal internalization of the full-length lPRLR. However, when the receptor is truncated (cT272), this region is able to mediate internalization at the same rate as the full-length lPRLR. The sPRLR contains only the first two of the dileucines in the second proximal region, suggesting roles in internalization of this isoform. The lack of the third dileucine (LL268/269) may also explain the slower internalization of the sPRLR compared with the lPRLR or cT272.

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Figure 8. Models for PRLR Internalization
The lPRLR has two regions that contain internalization motifs. The first region, containing a Phe residue (F290) and a nearby dileucine (LL286/287), is unique to the lPRLR and is essential for internalization of this isoform. F290 and LL286/287 cooperate and can independently contribute to internalization. The second region contains three dileucine motifs proximal to amino acid 272 and is also required for optimal internalization of the lPRLR. This region can effectively mediate internalization of the truncated receptor, cT272, when the sequence distal to amino acid 272 is removed, and individual dileucines cooperate in directing internalization of cT272. The sPRLR contains only the first two proximal dileucines and may utilize these for internalization. The lack of the third dileucine in the sPRLR may also explain the slower internalization of this receptor compared with the lPRLR and cT272. Nevertheless, despite the slower rate, these dileucines are able to mediate internalization of the sPRLR through similar endocytic pathways as the lPRLR.
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The internalization motifs identified in the lPRLR are conserved among all the lPRLRs characterized to date. A Phe residue or the conservative substitute Tyr and nearby dileucine in the first region are found in similar positions, suggesting that this region is critical for ligand-induced endocytosis across species (Fig. 1
). The two distal dileucines in the second region are also highly conserved, consistent with a similar function. The second dileucine (LL259/260) in this region, shown here to be important for internalization of truncated receptor cT272 and possibly for the sPRLR as well, is also conserved in all the PRLRs and has been suggested as one of the internalization motifs for rat sPRLR (22). Internalization of the rat PRLR in a transfection system like that employed here occurred at rates very similar to that of bovine PRLR (22, 33, 34). Nevertheless, additional motifs may contribute to internalization of specific PRLR isoforms in some species. For example, a novel ß-turn (LPGG) in the unique C terminus of the rat sPRLR was also suggested to be an internalization motif (22). In contrast, the 11-amino acid unique tail of the bovine sPRLR did not contain either positive or negative elements (Fig. 3
). Different experimental systems (transient or stable transfection; cell type), as well as unique internalization motifs, particularly in the C termini of the sPRLR across species, may alter observed characteristics of the internalization process.
Two major categories of internalization motifs have been identified in membrane receptors: a Tyr-based motif (NPXY or YXX
;
represents hydrophobic residues such as L, I, M, V, F), and a dileucine motif (LL or IL) (for reviews, see Refs. 35 and 36). Many receptors possess multiple motifs for optimal internalization or different regulation, and mutation of a single motif often does not completely abolish receptor internalization (37, 38, 39). Several studies have suggested that they can function independently of one another (40, 41) and do not compete for endocytic machinery (42). Both the Tyr-based and dileucine motifs have been shown to bind to adaptor proteins (adaptor protein 2), which permit endocytosis through clathrin-coated pits (25, 39, 41, 43, 44, 45, 46, 47). Crystal structure studies have elucidated the interaction of the YXX
motif with the µ2 subunit of adaptor protein 2, and preference for the nonphosphorylated Tyr residue (43, 48, 49). A Phe residue, which is unable to be phosphorylated but is structurally similar to Tyr, appears to be an acceptable substitute for many membrane receptors, including the low-density lipoprotein receptor (50), cystic fibrosis transmembrane conductance regulator (39), and asialoglycoprotein receptor (51). Thus, the F290IEV (293) in the lPRLR may present a YXX
motif, which is consistent with either a Phe or Tyr in that position of lPRLRs across species.
Alternatively, the F290 may be part of an ubiquitin-dependent endocytosis motif, which has been shown to play a critical role in internalization of the full-length GHR. F290 of the lPRLR is in a context similar to F327 in the GHR, suggesting an analogous link to the ubiquitin-proteasome system. Indeed, mutating individual residues in the GHR UbE motif to those in the lPRLR did not affect utilization of this pathway, although not all residues were simultaneously altered (26). However, regardless of the function of the motif containing this residue in the lPRLR, it is clearly not as critical for internalization as for the GHR, since mutation inhibited lPRLR internalization only modestly in marked contrast to the GHR. These data suggest that accessibility or conformation of the Phe-containing motif in the lPRLR may be different from the GHR. The nearby dileucine (LL286/287) found in all PRLRs, but in none of the GHRs, may contribute to this. When the GHR was mutated by truncation to amino acid 349, LL347/348 signaled ubiquitin-independent endocytosis (52), and the F327, critical for the wild-type GHR, became less dominant (53). Nonetheless, in our parallel studies of PRLR and GHR endocytosis in the same experimental system, the lPRLR appeared to internalize about twice as rapidly as the GHR. Whether this is a result of utilization of different internalization motifs linked to different endocytic pathways, and/or a consequence of the multiple motifs employed by the lPRLR compared with the single dominant motif for the GHR, remains to be determined.
The GTPase dynamin is required for endocytosis through both the clathrin-mediated and caveolar pathways (28, 29, 30). Internalization of both the lPRLR and the sPRLR beyond that of the mutant cT234 (without a cytoplasmic domain) required dynamin. Thus, the signal(s) required for dynamin-mediated endocytic pathways may either be in the common region of the PRLR isoforms, or the different internalization motifs used by the two PRLR isoforms may be able to interact with the same machinery. Inhibition of internalization of PRLR isoforms by the clathrin Hub confirmed involvement of the clathrin-mediated pathway. These results are consistent with the report of ligand-dependent coimmunoprecipitation of the rat sPRLR with
-adaptin (22). However, they do not rule out involvement of other pathways, including caveolae.
Our studies have demonstrated that the lPRLR and sPRLR employ many of the same endocytic motifs, and are internalized via similar pathways, despite the difference in the apparent rate of this process. It remains to be determined whether these internalization motifs also play roles in directing subsequent trafficking and processing of the receptor once internalized. Further studies will be necessary to identify the mechanism(s) through which ligand stimulates this process, particularly in light of the marked differences in signaling capacities of the PRLR isoforms. Activation of JAK2, believed to be most proximal in the signaling pathway, does not appear to be required. Mutation of box 1 in the lPRLR isoform blocked association and consequently ability to activate JAK2, but did not alter internalization (data not shown and Ref. 34). These findings are consistent with those of the GHR, where the antagonists G120R and B2036 failed to activate JAK2, but permitted internalization similar to the wild-type receptor (54, 55). Direct examination of receptor trafficking will facilitate these studies.
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MATERIALS AND METHODS
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Plasmids and Hormones
The long and short isoforms of the bovine PRLR were cloned into the pcDNA3 expression vector (Invitrogen, San Diego, CA) under control of the cytomegalovirus promoter as previously reported (8, 56). Wild-type and K44E-dynamin-1 constructs were gifts from R. B. Vallee (Shrewsbury, MA; Ref. 28). Bovine clathrin Hub (T7-Hub) was a gift from F. M. Brodsky (San Francisco, CA; Ref. 31). Both dynamin and T7-Hub constructs were then subcloned into the pcDNA3 expression vector. Wild-type and F327A-GHR constructs were in the pcDNA3 vector as previously reported (19). Mutagenesis was performed according to the MORPH Site-Specific Plasmid DNA mutagenesis kit (5 Prime
3 Prime, Inc., Boulder, CO). Constructs were sequenced to confirm mutations. 125I-bPL and 125I-bPRL were prepared using Iodogen (57) by the Radionuclide Laboratory of the School of Veterinary Medicine, University of Wisconsin-Madison. Specific activity using this method was reproducibly about 30 µCi/µg. bPRL (USDA bPRL B-1, AFP 5300) was obtained from the USDA Animal Hormone Program of the USDA Reproduction Laboratory (Beltsville, MD), and recombinant bPL was a gift from the Monsanto Co. (St. Louis, MO). Previous work has shown that both bPL and bPRL bind the long and short isoforms of the bPRLR with about the same affinity [dissociation constant (Kd), 2.0 x 10-10 M], and that both are equally able to stimulate transcription from a PRL-responsive promoter (8, 56).
Cell Culture and Transient Transfections
COS-7 cells were grown in DMEM/Hams F12 medium (Sigma, St. Louis, MO), containing 5% heat-inactivated fetal bovine serum (HyClone Laboratories, Inc., Logan, UT), 100 U/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD) at 37 C in 5% CO2.
For studies examining internalization of the long, short, and mutated PRLR, 106 cells were transiently transfected by calcium phosphate precipitation as described previously (8, 56) or by SuperFect reagent (QIAGEN, Valencia, CA). The same results were obtained by either transfection method. For some experiments, a Rous sarcoma virus luciferase construct was cotransfected to monitor transfection efficiency. After transfection, cells were replated into 24-well plates (Costar, Cambridge, MA) at 24 x 105 cells per well depending on the experiment. Ligand binding was initiated 48 h after transfection.
Receptor Binding and Internalization
Transiently transfected cells were washed with 1 ml binding media (DMEM/F12 supplemented with 10 mM MgCl2; 1 mM CaCl2; 1% BSA; 1% penicillin-streptomycin, pH 7.6), followed by incubation in binding media for 40 min on ice to arrest internalization. Binding media were removed and 125I-bPL or -bPRL (150,000 cpm,
0.34 nM, near the Kd of these receptors), with or without a 300-fold excess (180 nM) unlabeled bPRL in 0.3 ml binding media was added per well. Cells were allowed to bind ligand for 20 h at 4 C. They were then washed three times with 1 ml ice-cold binding medium to remove unbound ligand. Binding medium (1 ml) was added and cells were moved to 37 C to initiate internalization, creating a pulse of ligand-bound cell surface receptors. After incubation at 37 C for 0 min to 2 h, cells were placed on ice and cell surface-bound material and internalized material were harvested. Cells were washed twice with 1 ml ice-cold binding media. Cell surface-bound ligand was harvested by acid treatment on ice: 0.5 ml of ice-cold 50 mM glycine (pH 2.5), 100 mM NaCl, was added to each well for 2 min and removed, followed by one wash with the same buffer and one with binding medium. These washes were pooled and radioactivity was determined. The internalized fraction was harvested by solubilization in 0.2 N NaOH, 1% sodium dodecyl sulfate, and counted. For some experiments, the media were analyzed for intact and degraded ligand by incubation in 10% trichloroacetic acid (final concentration) on ice for 30 min, and precipitable (intact) material was harvested by centrifugation at 2500 x g. Degraded ligand was determined as the difference between total radioactivity in the media, and precipitable radioactivity.
Specific binding in each fraction was determined as the difference between 125I-labeled ligand detected in the presence and in the absence of an excess of unlabeled hormone. The internalization ratio was expressed as a percentage of the specific internalized fraction with respect to total specific binding at each time point. The rate of change was calculated by determining separate regression lines whose slope defines the rate of change for each receptor isoform. Degradation was expressed as the percentage of degraded ligand to total specific binding at 0 min.
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FOOTNOTES
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This work was supported in part by NIH Grant R01 CA-78312 and the University of Wisconsin Center for Womens Health and Womens Health Research.
1 Current address: Department of Biochemistry/Molecular Biology, University of Minnesota-Duluth, Duluth, Minnesota 55812. 
Abbreviations: bPL, Bovine placental lactogen; bPRL, bovine prolactin; GHR, GH receptor; JAK, Janus kinase; lPRLR, long PRL receptor; PL, placental lactogen; PRL, prolactin; PRLR, PRL receptor; sPRLR, short PRL receptor; UbE, ubiquitin-dependent endocytosis.
Received for publication February 20, 2002.
Accepted for publication July 16, 2002.
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REFERENCES
|
---|
- Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA 1998 Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev 19:225268[Abstract/Free Full Text]
- Yu-Lee L, Luo G, Moutoussamy S, Finidori J 1998 Prolactin and growth hormone signal transduction in lymphohaemopoietic cells. Cell Mol Life Sci 54:10671075[CrossRef][Medline]
- Clevenger CV, Kline JB 2001 Prolactin receptor signal transduction. Lupus 10:706718[Medline]
- Bignon C, Daniel N, Belair L, Djiane J 1999 In vitro expression of long and short ovine prolactin receptors: activation of Jak2/STAT5 pathway is not sufficient to account for prolactin signal transduction to the ovine ß-lactoglobulin gene promoter. J Mol Endocrinol 23:125136[Abstract/Free Full Text]
- Ormandy CJ, Binart N, Helloco C, Kelly PA 1998 Mouse prolactin receptor gene: genomic organization reveals alternative promoter usage and generation of isoforms via alternative 3'-exon splicing. DNA Cell Biol 17:761770[Medline]
- Davis JA, Linzer DI 1989 Expression of multiple forms of the prolactin receptor in mouse liver. Mol Endocrinol 3:674680[Abstract]
- Anthony RV, Smith GW, Duong A, Prat SL, Smith MF 1995 Two forms of the prolactin receptor messenger ribonucleic acid are present in ovine fetal liver and adult ovary. Endocrine 3:291295
- Schuler LA, Nagel RJ, Gao J, Horseman ND, Kessler MA 1997 Prolactin receptor heterogeneity in bovine fetal and maternal tissues. Endocrinology 138:31873194[Abstract/Free Full Text]
- Chang W-P, Clevenger CV 1996 Modulation of growth factor receptor function by isoform heterodimerization. Proc Natl Acad Sci USA 93:59475952[Abstract/Free Full Text]
- Das R, Vonderhaar BK 1995 Transduction of prolactins (PRL) growth signal through both long and short forms of the PRL receptor. Mol Endocrinol 9:17501759[Abstract]
- Lesueur L, Edery M, Ali S, Paly J, Kelly PA, Djiane J 1991 Comparison of long and short forms of the prolactin receptor on prolactin-induced milk protein gene transcription. Proc Natl Acad Sci USA 88:824828[Abstract]
- ONeal KD, Yu-Lee LY 1994 Differential signal transduction of the short, Nb2, and long prolactin receptors. Activation of interferon regulatory factor-1 and cell proliferation. J Biol Chem 269:2607626082[Abstract/Free Full Text]
- Berlanga JJ, Garcia-Ruiz JP, Perrot-Applanat M, Kelly PA, Edery M 1997 The short form of the prolactin (PRL) receptor silences PRL induction of the ß-casein gene promoter. Mol Endocrinol 11:14491457[Abstract/Free Full Text]
- Duan WR, Linzer DIH, Gibori G 1996 Cloning and characterization of an ovarian-specific protein that associates with the short form of the prolactin receptor. J Biol Chem 271:1560215607[Abstract/Free Full Text]
- Nagano M, Kelly PA 1994 Tissue distribution and regulation of rat prolactin receptor gene expression. Quantitative analysis by polymerase chain reaction. J Biol Chem 269:1333713345[Abstract/Free Full Text]
- Boutin JM, Edery M, Shirota M, Jolicoeur C, Lesueur L, Ali S, Gould D, Djiane J, Kelly PA 1989 Identification of a cDNA encoding a long form of prolactin receptor in human hepatoma and breast cancer cells. Mol Endocrinol 3:14551461[Abstract]
- Hu Z-Z, Meng J, Dufau ML 2001 Isolation and characterization of two novel forms of the human prolactin receptor generated by alternative splicing of a newly identified exon 11. J Biol Chem 276:4108641094[Abstract/Free Full Text]
- Kline JB, Roehrs H, Clevenger CV 1999 Functional characterization of the intermediate isoform of the human prolactin receptor. J Biol Chem 274:3546135468[Abstract/Free Full Text]
- Govers R, van Kerkhof P, Schwartz AL, Strous GJ 1997 Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligand-induced internalization of the growth hormone receptor. EMBO J 16:48514858[Abstract/Free Full Text]
- Lobie PE, Sadir R, Graichen R, Mertani HC, Morel G 1999 Caveolar internalization of growth hormone. Exp Cell Res 246:4755[CrossRef][Medline]
- Strous GJ, Govers R 1999 The ubiquitin-proteasome system and endocytosis. J Cell Sci 112:14171423[Abstract/Free Full Text]
- Vincent V, Goffin V, Rozakis-Adcock M, Mornon J-P, Kelly PA 1997 Identification of cytoplasmic motifs required for short prolactin receptor internalization. J Biol Chem 272:70627068[Abstract/Free Full Text]
- Genty N, Paly J, Edery M, Kelly PA, Djiane J, Salesse R 1994 Endocytosis and degradation of prolactin and its receptor in Chinese hamster ovary cells stably transfected with prolactin receptor cDNA. Mol Cell Endocrinol 99:221228[CrossRef][Medline]
- Dittrich E, Haft CR, Muys L, Heinrich PC, Graeve L 1996 A di-leucine motif and an upstream serine in the interleukin- 6 (IL-6) signal transducer gp130 mediate ligand-induced endocytosis and down-regulation of the IL-6 receptor. J Biol Chem 271:54875494[Abstract/Free Full Text]
- Fan G-H, Yang W, Wang X-J, Qian Q, Richmond A 2001 Identification of a motif in the carboxyl terminus of CXCR2 that is involved in adaptin 2 binding and receptor internalization. Biochemistry 40:791800[CrossRef][Medline]
- Govers R, ten Broeke T, van Kerkhof P, Schwartz AL, Strous GJ 1999 Identification of a novel ubiquitin conjugation motif, required for ligand-induced internalization of the growth hormone receptor. EMBO J 18:2836[Abstract/Free Full Text]
- Allevato G, Billestrup N, Goujon L, Galsgaard ED, Norstedt G, Postel-Vinay MC, Kelly PA, Nielsen JH 1995 Identification of phenylalanine 346 in the rat growth hormone receptor as being critical for ligand-mediated internalization and down-regulation. J Biol Chem 270:1721017214[Abstract/Free Full Text]
- Herskovits JS, Burgess CC, Obar RA, Vallee RB 1993 Effects of mutant rat dynamin on endocytosis. J Cell Biol 122:565578[Abstract]
- Henley JR, Krueger EW, Oswald BJ, McNiven MA 1998 Dynamin-mediated internalization of caveolae. J Cell Biol 141:8599[Abstract/Free Full Text]
- Oh P, McIntosh DP, Schnitzer JE 1998 Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-dependent fission from the plasma membrane of endothelium. J Cell Biol 141:101114[Abstract/Free Full Text]
- Liu S-H, Marks MS, Brodsky FM 1998 A dominant-negative clathrin mutant differentially affects trafficking of molecules with distinct sorting motifs in the class II major histocompatibility complex (MHC) pathway. J Cell Biol 140:10231037[Abstract/Free Full Text]
- Barr VA, Lane K, Taylor SI 1999 Subcellular localization and internalization of the four human leptin receptor isoforms. J Biol Chem 274:2141621424[Abstract/Free Full Text]
- Perrot-Applanat M, Gualillo O, Buteau H, Edery M, Kelly PA 1997 Internalization of prolactin receptor and prolactin in transfected cells does not involve nuclear translocation. J Cell Sci 110:11231132[Abstract/Free Full Text]
- Perrot-Applanat M, Gualillo O, Pezet A, Vincent V, Edery M, Kelly PA 1997 Dominant negative and cooperative effects of mutant forms of prolactin receptor. Mol Endocrinol 11:10201032[Abstract/Free Full Text]
- Kirchhausen T 1999 Adaptors for clathrin-mediated traffic. Annu Rev Cell Dev Biol 15:732
- Keilker R, Spiess M, Crottet P 1999 Recognition of sorting signals by clathrin adaptors. Bioessays 21:558567[CrossRef][Medline]
- Kawakami K, Takeshita F, Puri RK 2001 Identification of distinct roles for a dileucine and a tyrosine internalization motif in the interleukin (IL)-13 binding component IL-13 receptor
2 chain. J Biol Chem 276:2511425120[Abstract/Free Full Text]
- Thomas WG, Baker KM, Motel TJ, Thekkumkara TJ 1995 Angiotensin II receptor endocytosis involves two distinct regions of the cytoplasmic tail. J Biol Chem 270:2215322159[Abstract/Free Full Text]
- Weixel KM, Bradbury NA 2000 The carboxyl terminus of the cystic fibrosis transmembrane conductance regulator binds to AP-2 clathrin adaptors. J Biol Chem 275:36553660[Abstract/Free Full Text]
- Li Y, Marzolo MP, van Kerkhof P, Strous GJ, Bu G 2000 The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein. J Biol Chem 275:1718717194[Abstract/Free Full Text]
- Rapoport I, Chen YC, Cupers P, Shoelson SE, Kirchhausen T 1998 Dileucine-based sorting signals bind to the ß chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site. EMBO J 17:21482155[Abstract/Free Full Text]
- Warren RA, Green FA, Stenberg PE, Enns CA 1998 Distinct saturable pathways for the endocytosis of different tyrosine motifs. J Biol Chem 273:1705617063[Abstract/Free Full Text]
- Boehm M, Bonifacino JS 2001 Adaptins, the final recount. Mol Biol Cell 12:29072920[Abstract/Free Full Text]
- Nesterov A, Carter RE, Sorkina T, Gill GN, Sorkin A 1999 Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant µ2 subunit and its effects on endocytosis. EMBO J 18:24892499[Abstract/Free Full Text]
- Bremnes T, Lauvrak V, Lindqvist B, Bakke O 1998 A region of the medium chain adaptor subunit (µ) recognizes leucine- and tyrosine-based sorting signals. J Biol Chem 273:86388645[Abstract/Free Full Text]
- Aguilar RC, Ohno H, Roche KW, Bonifacino JS 1997 Functional domain mapping of the clathrin-associated adaptor medium chains µ1 and µ2. J Biol Chem 43:2716027166[CrossRef]
- Hofmann MW, Honing S, Rodionov D, Dobberstein B, von Figura K, Bakke O 1999 The leucine-based sorting motifs in the cytoplasmic domain of the invariant chain are recognized by the clathrin adaptors AP1 and AP2 and their medium chains. J Biol Chem 274:3615336158[Abstract/Free Full Text]
- Owen DJ, Evans PR 1998 A structural explanation for the recognition of tyrosine-based endocytotic signals. Science 282:13271332[Abstract/Free Full Text]
- Marsh M, McMahon HT 1999 The structural era of endocytosis. Science 285:215219[Abstract/Free Full Text]
- Davis CG, van Driel IR, Russell DW, Brown MS, Goldstein JL 1987 The low density lipoprotein receptor. J Biol Chem 262:40754082[Abstract/Free Full Text]
- Geffen I, Fuhrer C, Leitinger B, Weiss M, Huggel K, Griffiths G, Spiess M 1993 Related signals for endocytosis and basolateral sorting of the asialoglycoprotein receptor. J Biol Chem 268:2077220777[Abstract/Free Full Text]
- Govers R, van Kerkhof P, Schwartz AL, Strous GJ 1998 Di-leucine-mediated internalization of ligand by a truncated growth hormone receptor is independent of the ubiquitin conjugation system. J Biol Chem 273:1642616433[Abstract/Free Full Text]
- van Kerkhof P, Alves dos Santos CM, Sachse M, Klumperman J, Bu G, Strous GJ 2001 Proteasome inhibitors block a late step in lysosomal transport of selected membrane but not soluble proteins. Mol Biol Cell 12:25562566[Abstract/Free Full Text]
- Harding PA, Wang XZ, Okada S, Chen WY, Wan W, Kopchick JJ 1996 Growth hormone (GH) and a GH antagonist promote GH receptor dimerization and internalization. J Biol Chem 271:67086712[Abstract/Free Full Text]
- Maamra M, Finidori J, Von Laue S, Simon S, Justice S, Webster J, Dower S, Ross R 1999 Studies with a growth hormone antagonist and dual-fluorescent confocal microscopy demonstrate that the full-length human growth hormone receptor, but not the truncated isoform, is very rapidly internalized independent of Jak2-Stat5 signaling. J Biol Chem 274:1479114798[Abstract/Free Full Text]
- Scott P, Kessler MA, Schuler LA 1992 Molecular cloning of the bovine prolactin receptor and distribution of prolactin and growth hormone receptor transcripts in fetal and utero-placental tissues. Mol Cell Endocrinol 89:4758[CrossRef][Medline]
- Miller WT, Speck JC 1983 Protein iodination using Iodogen. Int J Appl Radiat Isot 34:639641[Medline]