Deletions in the Cytoplasmic Domain of the Polymeric Immunoglobulin Receptor Differentially Affect Endocytotic Rate and Postendocytotic Traffic*

We have examined the function of the cytoplasmic domain of the polymeric immunoglobulin receptor (pig-R) by producing two separate deletions in the cytoplasmic domain of the pig-R, expressing the mu- tant receptors in polarized MDCK cells, and analyzing each for their effects on receptor and ligand traffic. Deletion of the C-terminal 30 amino acids (726-756) reduces the rate of internalization of receptor-bound ligand from the basolateral surface. However, this mutation has no effect on delivery of receptor from the Golgi to the basolateral surface or the post-endocytotic traffic of receptor and ligand. Mutation of a tyrosine at position 734 to serine produces a receptor with a similar phenotype. If residues 670-707 are deleted from the middle of the cytoplasmic domain, both baso- lateral delivery and internalization are unaffected. However, unlike wild type,

The polymeric immunoglobulin receptor (pig-R)' is responsible for the receptor-mediated transcytosis of dimeric IgA and pentameric IgM (polymeric immunoglobulins, pig) across various epithelia into external secretions. The itinerary of the pig-R begins with synthesis in the rough endoplasmic reticulum and processing in Golgi stacks, followed by delivery to *The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. !j Supported by National Institutes of Health Grant Kll HD00722. To whom correspondence should be addressed. Tel.:  11 Supported by National Institutes of Health Grant ROl-AI-21752.
the basolateral surface of polarized epithelial cells. At the basolateral surface, receptor binds pig, and the receptorligand complex is internalized via coated pits (Geuze et al., 1984). Receptor-ligand complexes pass through the endosomal compartment and are then delivered to the apical surface (Takahasi et al., 1982;Geuze et al., 1984). At or near the apical surface of the cell, receptor is cleaved to secretory component (SC), releasing pig with SC. Because of this complex sorting pathway, the receptor mediated transcytosis of pig serves as a excellent model system for the study of receptor and ligand traffic in polarized epithelial cells. In order to examine the factors that control this traffic, we have expressed the cDNA for rabbit pig-R in Madin-Darby canine kidney (MDCK) cells by utilizing a retroviral expression vector . MDCK cells are cultured on suspended permeable filters such that the basolateral and apical domains are biochemically separate. Using this system, we have shown that the biosynthetic and transcytotic pathways of the pig-R mimic that found in uivo .
We have suggested that the cytoplasmic domain of the pig-R contains structural determinants or signals that play a role in receptor and ligand traffic since deletion of the entire cytoplasmic domain of the pig-R (tail-minus mutant) prevents both basolateral delivery of newly synthesized receptor and internalization . A role for the cytoplasmic domain in the intracellular traffic of several other integral membrane proteins has been proposed. For example, Gonzalez and co-workers (1987) have suggested an important role for the cytoplasmic domain of the vesicular stomatitis virus G-protein in the delivery of the newly synthesized protein to the basolateral surface of epithelial cells. The cytoplasmic domains for the low density lipoprotein (LDL) receptor (Lehrman et al., 1985), transferrin receptor (Rothenberger et al., 1987), and epidermal growth factor receptor (Schlessinger, 1986) have been demonstrated to be important for internalization.
Interestingly, point mutations of the tyrosine residue(s) within the cytoplasmic domains of the LDL receptor (Davis et al., 1987), the transferrin receptor (Jing et al., 1990) Breitfeld et al., 1989a) or the two pig-R mutations described in Fig. 1 were used for study.

Construction
of Mutant DIP-R Clones-All recombinant DNA manipulations utilized standard-techniques (Maniatis et al., 1982). The replicative form of the Ml3 Mp8 cloning vector containing the coding region of the pig-R with BglII sites at the ends of the coding region Breitfeld et al., 1989a)  Expression of Mutant cDNA pig-R in MDCK Cells-As described previously Breitfeld et al., 1989a), the two mutant pig-R constructions were separately ligated into a retroviral vector (pWE) and with the use of the $ packaging system (Cone and Mulligan, 1984)  The incubation was continued at 37 "C! for the designated chase period. At the end of the chase period, cells as well as the apical and basolateral media were harvested and immunoprecipitated with guinea pig anti-rabbit SC antibody, as described previously (Breitfeld et al., 1989a At the end of the chase period, cells were harvested and immunoprecipitated as described above. Zodination of An&-SC-Fab Fragments and dZgA-The generation of affinity purified guinea pig anti-rabbit SC Fab fraements has been described previously (Breitfeld et al., 1989a). The anti-SC Fab fragments and human dIgA (gift of Dr. M. Schiff, University of Toronto) were iodinated by the iodine monochloride method (Goldstein et al., 1983)

RESULTS
The wild-type pig-R is a 755-amino acid polypeptide chain which contains one putative membrane spanning domain of 23 amino acids and a C-terminal cytoplasmic domain of 103 amino acids, residues 653-755 . We have shown previously that when 101 C-terminal amino acids of the cytoplasmic domain are deleted (tail-minus mutant), the mutant receptor expressed in MDCK cells not only fails to internalize ligand but is delivered after biosynthesis to the apical surface rather than the basolateral surface . We have now further investigated the function of the cytoplasmic domain of the pig-R by constructing two deletion mutations within the cytoplasmic domain of the pig-R. We describe their expression and functional analysis in this report. These mutations are summarized in Fig. 1.

Expression and Biosynthesis of p&-R Mutants in MDCK
Cells-Using a retroviral vector and the # packaging system Wild-Type as described previously Breitfeld et al., 1989a), both the wild-type and mutant pig-Rs were expressed in MDCK cells. To examine the biosynthesis of the mutant pig-Rs, MDCK cells expressing the wild-type pig-R or a mutant pig-R were pulse-labeled with [35S]cysteine for 20 min at 37 "C and chased for various periods of time. Cells, as well as apical and basolateral media, were harvested, immunoprecipitated with anti-SC antibody, and analyzed by SDS-PAGE and fluorography. Fig. 2A demonstrates (as reported previously) that the wild-type pig-R is first synthesized as a 90-kDa polypeptide (lane 1) that is modified by the addition of complex carbohydrates to a doublet of 100 and 105 kDa during the subsequent chase period (lanes 2-5). This mature form of the pig-R is cleaved to SC and released into the apical medium (lanes 8, 10, and 12). Fig. 2B displays the pulse-chase analysis of the 725t pig-R. The results are similar to those found with wild-type receptor except that the 725t pig-R migrates faster on SDS-PAGE, since 30 amino acids have been deleted from the cytoplasmic domain. In particular, like wild-type receptor, after the mature product is formed, SC is released into the apical medium (  sized as a precursor (lane I) which is converted to a doublet of 88 and 90 kDa by 1 h of chase (lane 2), reflecting processing of its oligosaccharide side chains to the complex type in the Golgi. Much like wild-type receptor, this newly synthesized A670-707 pig-R is degraded during the subsequent chase period (lanes 3-5), but, unlike wild-type, no SC is released into the apical medium (lanes 6, 8, 10, and 12). Rather, the A670-707 pig-R is apparently degraded to products that are too small to be detected by our immunoprecipitation/SDS-PAGE protocol.
To further characterize the degradation of the A670-707 pig-R, we asked if the presence of ligand would have an effect on this degradation. Ligand binding can alter the fate of several other receptors resulting in receptor degradation in acidic compartments such as late endosomes and lysosomes (Stoscheck and Carpenter, 1984). Therefore, cells expressing the A670-707 pig-R were metabolically labeled and chased for various times, either in the absence (Fig. 3A) or presence of ligand (dIgA at 10 pg/ml, Fig. 3B). The cells and media were harvested and immunoprecipitated with anti-SC antibody. We determined the rate of A670-707 pig-R degradation by laser densitometry of the immunoprecipitated bands. Fig.  3A demonstrates that the A670-707 pig-R is rapidly degraded in MDCK cells with a half-life of approximately 1 h. At 4 h of chase, less than 10% of the A670-707 pig-R remains in cells (Fig. 3A, lane 4), yet there is no SC released into the medium (lanes 5 and 6). Fig. 3B demonstrates that added ligand has very little effect on the rate of A670-707 pig-R degradation (compare with Fig. 3A). As in control A670-707 pig-R cells, no SC is released into the medium after 4 h of chase (lanes 5 and 6, compare Fig. 3, A and B). This suggests that the degradation of newly synthesized A670-707 pig-R is independent of ligand.

Biosynthetic
Delivery of Wild-type and Mutant pig-Rs-To determine whether newly synthesized receptor is delivered to either the apical or basolateral surface of MDCK cells, a trypsin assay was developed. This assay was modeled after that utilized by Matlin and Simons (1984) to examine surface delivery of influenza virus hemagglutinin in MDCK cells. Our assay conditions were optimized with MDCK cells which express the wild-type pig-R, since we had shown previously, by an antibody-dependent assay, that over 90% of newly synthesized wild-type pig-R is delivered first to the basolat-era1 surface ). We could not use this earlier assay for the A670-707 pig-R in particular since the antibody-dependent assay requires cleavage of the receptor to SC at the apical surface. Therefore, MDCK cells expressing either wild-type or mutant pig-R were pulse-labeled with [35S]cysteine for 10 min and chased for 45 min in the absence or presence of 25 pg/ml trypsin in the basolateral or apical medium. Soybean trypsin inhibitor at 200 rg/ml was included in the medium opposite to trypsin. The entire pulse and chase periods were performed at 37 "C. Newly synthesized receptor molecules which reach the surface exposed to trypsin will be digested, resulting in a decrease compared to control in the amount of immunoprecipitable protein. Fig. 4A demonstrates the results for such an experiment with the wild-type pig-R. After the pulse period, the pig-R precursor is identified (lane I). After the chase period of 45 min, the mature form of the receptor is identified (lane 2). If trypsin is included in the basolateral medium during the chase period, all newly synthesized receptor is digested (he 3). Yet, if trypsin is included in the apical medium during the chase, little or no receptor is digested (lane 4). These results are entirely consistent with those found previously  and confirms that newly synthesized wildtype pig-R is delivered to the basolateral surface. In addition, the incubation with apical trypsin (lane 4) suggests that, under the conditions of our assay, apical trypsin is not entering the cellular compartments responsible for the delivery of newly synthesized receptor to the basolateral surface and not leaking across the monolayer to the basolateral surface. Moreover, we found that the tail-minus mutant, which lacks 101 residues of the cytoplasmic domain and is not basolaterally targeted, is not digested by adding trypsin to the basolateral medium (Casanova et al., 1990).
An identical experiment was performed with MDCK cells which express either the 725t pig-R (Fig. 4B) or the A670-707 pig-R (Fig. 4C). As with wild-type receptor, each of these pig-R mutants is delivered to the basolateral surface, since exposure of cells to basolateral trypsin during the chase period digests newly synthesized mutant pig-Rs (lane 3, B and C). None reaches the apical surface during the 45 min of chase, since the addition of apical typsin has no effect (lane 4, B and C). This data demonstrates that the wild-type pig-R as well as the mutant pig-Rs are vectorally delivered to the basolat-era1 surface of MDCK cells after biosynthesis.
In a similar fashion, we examined the rate of delivery of newly synthesized wild-type and mutant receptor to the basolateral surface. After metabolic labeling, cells were chased for various periods of time without or with trypsin in the basolateral compartment.
After immunoprecipitation and SDS-PAGE, the amount of receptor appearing at the basolateral surface was quantitated by laser densitometry. Fig. 5 demonstrates that 90% of wild-type pig-R reaches the basolateral surface by 45 min and the half-time for this process is approximately 25-30 min. The 725t pig-R appears at the basolateral surface with a slightly shorter half-time (no longer than 15 min) compared with wild-type, whereas the A670-707 pig-R follows roughly the same time course as wild-type receptor. Thus, we conclude that amino acid segments 670-707 and 726-755 are not required for delivery of newly synthesized receptor to the basolateral surface, although deletion of residues 726-755 may enhance the rate of transport to the surface. Of particular note, almost all newly synthesized A670-707 pig-R reaches the basolateral surface within the 45-min chase period and, thus, prior to its intracellular degradation.
Since we have shown previously that deletion of 101 amino acids of the 103 amino acid cytoplasmic domain of the pig-R prevents proper delivery of newly synthesized receptor to the basolateral surface , we suggest that amino acids 653-669 and/or 708-725 are needed for this event.

Time Course of Internalization of Receptor-bound
Liand-To quantitate internalization of receptor-bound ligand from the basolateral surface, MDCK cells which express wild-type or mutant pig-R were allowed to bind radioiodinated Fab fragments derived from a polyclonal antibody raised against rabbit SC (anti-SC Fab fragments) at the basolateral surface for 2 h at 4 "C. We have shown previously that anti-SC Fab fragments function as native ligand (dIgA) (Breitfeld et al., 1989b). This substitution was required since radioiodinated dIgA, when utilized for basolateral binding assays, produces an unacceptable level of background binding at 4 "C as a consequence of nonspecific binding to the filter on which cells are cultured (Breitfeld et al., 1989b). After extensive washing, cells were rapidly warmed to 37 "C for various periods of time up to 12 min. After the designated warm-up period, cells were rapidly cooled to 4 "C and exposed to proteolysis at the basolateral surface with both chymotrypsin and proteinase K at 50 rg/ml for 60 min at 4 "C. This proteolysis step efficiently strips the basolateral surface of cell surface bound noninternalized anti-SC Fab fragments, yet does not disrupt the MDCK cell monolayer (Breitfeld et al., 1989b). The amount of radioactivity was then determined in the basolateral warm-up medium, the protease-sensitive fraction (surface), and protease-resistant fraction (intracellular) .
The percentage of ligand internalized as a function of time for wild-type and mutant pig-R is displayed in Fig. 6. For wild-type receptor, approximately 80% of bound anti-SC Fab fragments are internalized from the basolateral surface. This process is rapid, having a half-time of less than 1 min and reaching a maximum by 2.5 min. At 12 min, there is a decrease in the amount of intracellular anti-SC Fab fragments. This  ) for 2 h at 4 "C. and the basolateral surface was extensively washed.
The cells were rapidly warmed to 37 "C for the times indicated (1-12 min) and ranidlv cooled to 4 "C. The basolateral medium was harvested, and the ha&lateral surface was exposed to a mixture of 50 pg/ml chymotrypsin and 50 pg/ml proteinase K for 60 min at 4 "C. After washing, the protease-sensitive fractions were pooled and the cells were harvested.
The amount of radioactivity was determined in the basolateral medium (medium), the protease-sensitive fraction (cell surface), and the cells ( represents return of a fraction of basolaterally internalized ligand to the basolateral medium, as shown previously (Breitfeld et al., 1989b).
The A670-707 pig-R also rapidly internalizes anti-SC Fab fragments, reaching a maximum at 5 min with a half-time of less than 1 min. Thus, the time course of internalization for the A670-707 pig-R is similar to that of wild-type receptor, although the A670-707 pig-R is slightly slower. This suggests that the 38-amino acid deletion from 670 to 707 is not critical for the rapid internalization of receptor-bound ligand.
In contrast, the 725t pig-R displayed a slower rate of internalization of ligand compared with wild-type or A670-707 pig-R (see Fig. 6). The half-time for internalization for the 725t pig-R was approximately 2.5 min (40% of wild-type receptor). By comparison, the tail-minus receptor, as described previously , demonstrates no detectable internalization of ligand in this assay (see Fig. 6). (We observed an appreciable amount of anti-SC Fab binding to the basolateral surface of MDCK cells which express the tail-minus receptor. Although approximately 90% of newly synthesized tail-minus receptor is delivered to the apical surface, 10% is delivered basolaterally. Since this receptor has no detectable internalization, the tail-minus receptor accumulates at the basolateral surface, accounting for the high level of ligand binding.) Thus, the cytoplasmic domain of the pig-R is critical for internalization of ligand, and the results obtained for 725t pig-R demonstrate that rapid internalization (but not all internalization) is impaired by deletion of the C-terminal 30 amino acids. However, neither delivery of newly synthesized receptor to the basolateral surface nor postendocytotic delivery of ligand was affected by the 725t pig-R mutation (see below).
It should be noted that a cytoplasmic tyrosine is contained within the deleted segment of the 725t pig-R (Tyr-734). Since mutation of tyrosine residues in both the LDL receptor (Davis et al., 1987) and the mannose 6-phosphate receptor (Lobe1 et al., 1989) impairs the rapid internalization of ligand, we asked if Tyr-734 might be similarly involved in internalization. Using oligonucleotide-directed mutagenesis, we changed the tyrosine at position 734 to serine, producing a receptor termed Ser-734 pig-R. As indicated in Fig. 6, the time course of internalization for the Ser-734 pig-R is identical to the slow rate observed for 725t pig-R. In all other assays, the Ser-734 pig-R behaved as the 725t pig-R (see Figs. 2,4,5,7, and 8 (data not shown)).

Post-endocytotic
Delivery of Ligand-We have shown previously that, by using the basolateral proteolysis assay described above, a single cohort of internalized anti-SC Fab fragments can be isolated and its fate after endocytosis followed in MDCK cells that express the wild-type receptor (Breitfeld et al., 1989b). Using this assay, we have shown that anti-Fab fragments are transcytosed, but a significant fraction (45%) recycles to the basolateral medium. This assay involves several cooling steps and a protease treatment of the basolat-era1 surface prior to the final incubation at 37 "C. In order to develop a simpler assay of ligand delivery after endocytosis, we have modified the ligand uptake assay developed by Goldstein and co-workers (1983) for the LDL-receptor system. MDCK cells expressing wild-type or mutant pig-R were allowed to endocytose ligand for a brief time (10 min) at 37 "C, washed quickly to remove nonendocytosed ligand, and then apical and basolateral medium samples were examined for the appearance of either intact or degraded ligand over the 120min incubation. All steps were performed at 37 "C without any intervening cooling steps. Protease stripping of uninternalized ligand was not found to be necessary. Fig. 7A  onstrates that over 120 min, for wild-type receptor, 53% of endocytosed anti-SC Fab fragments is transcytosed, 20% returns to the basolateral medium, and only 6% is degraded and released into the apical and basolateral medium. The balance (21%) remains cell-associated after 120 min. Fig. 7B demonstrates the results of such an experiment with 725t pig-R and indicates that the fate of anti-SC Fab fragments endocytosed by the 725t pig-R is similar to wild-type receptor. However, the fate of ligand endocytosed by the A670-707 pig-R is quite different from wild-type (Fig. 7C). Only 7% of ligand is transcytosed into the apical medium intact. This represents 13% of the amount of transcytosis observed for wild-type receptor. Whereas 6% of ligand endocytosed by wild-type receptor is degraded and released into the medium, 50% of ligand endocytosed by the A670-707 pig-R is degraded and released (Fig. 7C). This degradation represents an 8-fold increase over that found with wild-type receptor. Like wildtype, 24% of ligand recycles to the basolateral medium intact. The balance (19%) remains cell associated after 120 min. Thus, post-endocytotic delivery of anti-SC Fab fragments is substantially altered when endocytosed by the A670-707 pig-R with a shift from the transcytotic pathway to one of degradation.
Thus, deletion of residues 670-707 affects ligand delivery after endocytosis but does not affect basolateral delivery of receptor or the time course of internalization of receptor-bound ligand. Ligand Dissociation-Are the altered phenotypes observed for our mutant pig-Rs due to alterations in receptor traffic produced by the mutations or simply due to altered rates of ligand dissociation from mutant receptor? For example, ligand may dissociate more rapidly from the 725t pig-R, and thus an apparant altered time course of internalization would be observed. Alternatively, internalized ligand may dissociate more rapidly from the A670-707 pig-R (especially in acidic intracellular compartments) and then ligand may be delivered by fluid phase to lysosomes for degradation.
To address these issues, we measured the time course of dissociation of ligand (anti-SC Fab fragments) at 37 "C from the basolateral surface of cells expressing either wild-type or mutant receptor. To inhibit endocytosis of receptor-bound ligand we included 0.2 M sucrose in the medium. As observed by others (Heuser and Anderson, 1989), we found in preliminary experiments that this hypertonic treatment inhibited at least 85% of anti-SC Fab fragment internalization by wild-type or mutant pig-R. The time course of dissociation of anti-SC Fab fragments from receptor is virtually the same for wild-type and mutant pig-Rs (Fig. 8A).
If the mutant pig-Rs are grossly altered in structure, the rate of dissociation of the natural ligand (dIgA) might differ from wild-type.
Therefore, we also investigated the time course of dissociation of dIgA from wild-type and mutant receptors. We found that the time course for dIgA dissociation was very similar for all forms of the receptor (Fig. 8B), suggesting that the structure of the ligand binding domain is not altered by any of our mutations.
Of note, under these conditions, the rates of dissociation for anti-SC Fab fragments are substantially slower than for dIgA. Since ligand might also dissociate intracellularly, particularly in acidic endosomes, we also measured rates of dissociation of ligand from receptor at pH 5.3 (which also inhibits endocytosis (Cosson et al., 1989)). At acidic pH, dissociation rates were similar for wild-type and mutant receptors with both anti-SC Fab fragments (Fig. 8C) and dIgA (Fig. 80).
Thus, the altered time course of ligand internalization demonstrated for the 725t pig-R and the altered fate of ligand endocytosed by the A670-707 pig-R is unlikely to be the consequence of altered rates of dissociation of ligand from receptor.
Since ligand (either anti-SC Fab fragments or dIgA) does dissociate from receptor at an appreciable rate at 37 "C, determining the fate of the ligand is an imperfect way to follow the fate of the receptor. However, it is unlikely that the ligand is simply entering the fluid phase after endocytosis. First, the majority of a fluid phase marker endocytosed at the basolateral surface of MDCK cells is delivered to lysosomes (70%) with only 10% transcytosed to the apical surface (Bom-se1 et al., 1989). Yet we find that very little anti-SC Fab fragments are degraded after endocytosis by wild-type receptor. Second, the post-endocytotic delivery of ligand is altered by mutations of the pig-R. If anti-SC Fab fragments were simply entering the fluid phase, one would not expect that receptor structure would affect delivery of ligand.

DISCUSSION
The intracellular traffic of receptors and ligands is a central issue in cell biology. Previously we found that deleting 101 of the 103 amino acids of the cytoplasmic domain of the pig-R 3) (c and D). Ligand (either ""I-anti-SC Fab fragments (A and C) or lZ51-dIgA (B and D)) was bound to the basolateral surface for 2 h at 4 "C and then the basolateral surface was washed extensively. Cells were warmed to 37 "C, and the basolateral medium was sampled at the times indicated. Ligand released was determined by y counting. MDCK cells that do not express the plg-R were analyzed in parallel, and these values were subtracted as nonspecific background. All points are the mean of duplicate filters. interfered with both basolateral delivery and with internalization of the receptor. In the current study we have found that deleting the C-terminal 30 residues yields a receptor which is internalized slowly, but otherwise follows the same pathway as wild-type receptor. A deletion of 38 residues from the middle of the cytoplasmic domain yields a receptor which is delivered to the basolateral surface and internalized almost as rapidly as wild-type receptor, but then is largely degraded instead of transcytosed. We have therefore demonstrated that different segments of the cytoplasmic domain of the pig-R, when deleted, affect different types of receptor and ligand traffic in polarized cells.

One explanation
for these results is that these mutations grossly perturb the folding of the receptor, leading to aberrant traffic. The definitive method to investigate protein folding is x-ray crystallography.
Other methods that have been used include binding of a panel of well defined monoclonal antibodies that are conformational specific (Copeland et al., 1988), sedimentation in sucrose gradients (Ng et ul., 1989), changes in resistance to proteolysis and/or denaturation (Doms and Helenius, 1986), or various assays of function of the protein (Schlessinger, 1986). Since we can quantitatively assay several functions of the pig-R, we have used the latter approach. Unlike several other membrane proteins whose mutants may not reach the cell surface because they are not folded properly (e.g. HA and vesicular stomatitis virus G-protein (reviewed in Rose and Doms, 1988)), all of our mutant pig-Rs are rapidly and accurately delivered to the (basolateral) surface. Moreover, all bind ligand (both anti-SC Fab fragments and dIgA) and the rates of dissociation of ligand (at pH 7.4 and 5.3) are very similar to wild-type.
By these functional criteria, the mutant pig-Rs appear to be folded correctly.
For the A670-707 pig-R, misfolding could lead to artifactual aggregation or oligomerization and thus cause mistargeting to lysosomes. Cross-linking of other receptors by antibodies can cause such mistargeting (Mellman and Plutner, 1984). However, we have shown that cross-linking the wild-type pig-R with polyvalent antibodies against the receptor do not prevent it from being transcytosed . Similarly, large polyvalent antigen-antibody complexes are also transcytosed by the pig-R . Finally, we have been unable to detect oligomers of the wild-type or mutant pig-R's by chemical cross-linking or by sucrose gradients at neutral and mildly acidic pH values. Thus, we think it is unlikely that our mutations have caused aberrant aggregation of the receptor.
Our most striking finding is that different mutations in the cytoplasmic domain can produce different phenotypes. If these mutations acted by grossly altering the folding of the receptor, then at least three different artifactual conformations of the receptor must exist. One would correspond to the previously reported tail-minus receptor which is not basolaterally delivered, whereas the other conformations would correspond to the slow internalization and degradation phenotypes reported here.
Taken together these arguements make it unlikely that our results are simply due to misfolding of the receptor. Another possible explanation is that the cytoplasmic domain of the pig-R contains several structural determinants which act as independent sorting signals and may interact with proteins in the cytoplasm. This hypothesis can be tested by transferring the putative sorting signals to other proteins.
What putative sorting signal could be deleted in the A670-707 pig-R mutant? The wild-type pig-R very efficiently avoids intracellular degradation.
Perhaps the cytoplasmic domain of the pig-R contains a signal for avoiding such degradation, and the A670-707 mutation has directly or indirectly disrupted this signal. Such a lysosomal avoidance signal has not been suspected previously and may be present on other normally recycling receptors (e.g. for asialoglycoproteins).
Another possibility is that the mutation has created a lysosomal targeting signal in the cytoplasmic domain.
The 725t pig-R (and Ser-734 pig-R) internalizes ligand slowly with the initial rate of internalization being no more than 40% of control. This rate is still far greater than that of the tail-minus receptor which completely blocks internaliza-tion. This suggests that the C-terminal 30 amino acids (especially tyrosine 734) play an important role in the internalization step but that the cytoplasmic domain of the pig-R may contain more than one signal for internalization.
In fact the pig-R cytoplasmic domain contains a second tyrosine at residue 668. However, mutation of only that residue to serine has no effect on internalization. ' In the case of the slow internalization mutants, the putative signal may be related to the tyrosine signal for internalization found in the LDL and mannose 6-phosphate receptors. It should be noted that for the LDL receptor, mutation of a cytoplasmic tyrosine decreases the internalization index to 30% of control (Davis et al., 1987) but does not completely block endocytosis of the receptor, similar to our results with the pig-R. However, the general importance of cytoplasmic tyrosine residues is illustrated by the observation that a tyrosine residue added to the cytoplasmic domain of a protein which is normally excluded from coated pits can induce its internalization via coated pits (Lazarovits and Roth, 1988). Finally, we have found that if the entire cytoplasmic domain is deleted, the receptor is apically delivered, whereas if residues 670-707 or 726-755 are deleted, the receptor is basolaterally delivered. This suggests that either residues 653-669 and/or 708-725 are involved in basolateral delivery.