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. 2000 Aug;11(8):2643-55.
doi: 10.1091/mbc.11.8.2643.

Down-regulation of cell surface receptors is modulated by polar residues within the transmembrane domain

L Zaliauskiene  1 , S Kang, C G Brouillette, J Lebowitz, R B Arani, J F Collawn
Affiliations
Free PMC article

Down-regulation of cell surface receptors is modulated by polar residues within the transmembrane domain

L Zaliauskiene et al. Mol Biol Cell. 2000 Aug.
Free PMC article

Abstract

How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the "signal" within the TM necessary and sufficient for down-regulation is Thr(11)Gln(17)Thr(19) (numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well ( approximately 79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals.

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Figures

Figure 1
Figure 1
(A) Schematic diagram of the TRTM mutants. A schematic diagram is shown of the MHC class II Ii showing the wild-type TM sequence. The IiTM 11–19 chimera contains a nine-amino acid substitution (shaded area) from the Ii that promotes lysosomal targeting (Kang et al., 1998). All other portions of this chimera are derived from the wild-type TR (unshaded areas). For each of the TM mutants prepared, the amino acid substitutions are shown. Constructs are referred to throughout by the corresponding names shown on the left. (B) Structural models for the TR dimeric TM. Left, three-dimensional ribbon model of the TM of the TRTM Thr11Gln17Thr19 mutant, represented as a helical dimer with the carboxy-terminus at the top. Only side chains of polar residues are depicted. The numbering system shown is the same as that used in Figure 1. Right, helical wheel representation of the same dimer, viewed from the carboxy-terminal end of the helix. Only polar residues are depicted. The software program Ribbons 3.1 was used (Carson, 1997).
Figure 2
Figure 2
Polar residues in the TR TM are necessary and sufficient to mediate targeting of the TR to a post-Golgi–processing compartment. Equivalent cell numbers of CEFs expressing wild-type TR, IiTM 11–19, TRTM Thr11Gln17Thr19, and IiTM 11–19 Ala11Ala17Ala19 chimeras were pulse labeled for 30 min with trans-35S label and chased with complete medium for various periods of time as indicated. Wild-type or mutant TRs were then immunoprecipitated from postnuclear supernatants and analyzed on SDS-polyacrylamide gels (A) as described under MATERIALS AND METHODS. Dried gels were exposed to XAR film overnight (Kodak). Radioactivity in the bands was quantitated on a PhorphorImager (B). A representative experiment of four is shown.
Figure 3
Figure 3
Colocalization of wild-type and mutant TR with LEP100. CEF expressing wild-type TR (A), TRTM Thr11Gln17Thr19 (B), IiTM 11–19 (C), and IiTM 11–19 Ala11Ala17Ala19 (D) were fixed, permeabilized, and stained with rabbit anti-TR antisera followed by Oregon Green-labeled goat anti-rabbit Ig or mouse anti-LEP100 followed by Texas Red-labeled goat anti-mouse IgG. Colocalization of the two proteins is indicated by yellow fluorescence.
Figure 4
Figure 4
Comparison of internalization rates of wild-type TR and various TR TM mutants. Equivalent numbers of CEFs expressing wild-type TR and TRTM Thr11Gln17Thr19 (A) or IiTM 11–19 and IiTM 11–19 Ala11Ala17Ala19 chimeras (B) were incubated with prewarmed (37°C) 125I-labeled Tf for indicated times. The amounts of internalized (internal Tf) and surface associated (surface Tf) radiolabel were determined as described under MATERIALS AND METHODS. Data are plotted with IN/SUR method, in which the slope of the line equals the endocytic rate constant ke (Wiley and Cunningham, 1982). The data represent the mean ± SE from five experiments for each time point.
Figure 5
Figure 5
Degradation of Tf bound to wild-type TR and TR TM mutants. Equivalent cell numbers of CEFs expressing wild-type TR, IiTM 11–19, TRTM Thr11Gln17Thr19, and IiTM 11–19 Ala11Ala17Ala19 were preincubated in serum-free medium for 30 min at 37°C and then incubated with 125I-labeled Tf in BSA-PBS for 10 min. at 37°C. The cells were then washed and reincubated at 37°C in DMEM containing 50 μg/ml unlabeled Tf and 0.1% BSA for various times. Acid-soluble radioactivity (○) or acid-insoluble 125I-labeled Tf (▵) released into the medium, as well as surface bound Tf (acid, □) and internalized (base, ⋄) 125I-labeled Tf was determined as described under MATERIALS AND METHODS and is expressed as a percentage of total radioactivity recovered. Each data point is the mean ± SE from an experiment done in triplicate. A representative experiment of three is shown.
Figure 6
Figure 6
Sucrose gradient sedimentation of TR TM mutants. CEF cells expressing wild-type TR, TRTM Thr11Gln17Thr19, IiTM 11–19, and IiTM 11–19 Ala11Ala17Ala19 were pulse labeled with trans-35S label for 2 h and chased 1 h in complete medium. Lysates were loaded onto 5–25% sucrose gradients and centrifuged as described under MATERIALS AND METHODS. Fractions were collected (numbering is from the top of the gradient), immunoprecipitated with anti-TR antibody, and analyzed by SDS-PAGE. Dried gels were exposed to XAR film (Kodak) overnight. Immunoprecipitates were quantitated on a PhosphorImager and the relative amounts in each fraction are shown above. The positions of molecular weight standards are shown: A, aldolase (7.4S); B, catalase (11.3S); and C, thyroglobulin (19.3S). A representative experiment of three is shown.
Figure 7
Figure 7
Analysis of TR TM mutants by chemical cross-linking. CEF cells expressing wild-type TR, TRTM Thr11Gln17Thr19, IiTM 11–19, and IiTM 11–19 Ala11Ala17Ala19 were pulse labeled with trans-35S label overnight and chased in complete medium for 2 h. Cells were lysed in 1% Triton X-100 and the cell lysates were cross-linked with 1 mM DTSSP for 20 min at room temperature. Receptors were isolated by immunoprecipitation, analyzed by SDS-PAGE under nonreducing conditions, and quantitated on a Phosphoimager. Bracketed numbers indicate the percentage of total protein present in each fraction. A representative experiment of three is shown.
Figure 8
Figure 8
Degradation of Tf and cross-linked Tf bound to the wild-type TR. CEFs expressing wild-type TR were preincubated in serum-free medium for 30 min at 37°C and then incubated with 125I-labeled Tf or 125I-labeled cross-linked Tf for 10 min. at 37°C. The cells were then washed and reincubated at 37°C in DMEM containing 50 μg/ml unlabeled Tf and 0.1% BSA for various times. Acid-soluble radioactivity (○) or acid-insoluble 125I-labeled Tf (▵) released into the medium, as well as surface bound Tf (□) and internalized (⋄) 125I-labeled Tf was determined as described in Figure 5. A representative experiment of three is shown.
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