Schematic representation of TβRII, TβRI, and their mutants. Myc- or HA-tagged TβRII or TβRI in pcDNA3 or pcDNA1 were prepared as described (Ref. 33 and “Experimental Procedures”). The numbers to the right of the truncation mutants indicate the last amino acid residue after which the stop codon was inserted. Dashed lines indicate deletions. The numbers after the Ala substitutions are those of the residues replaced by Ala. A, human TβRII (for sequence, see Ref. 9) consists of 565 amino acids. The EC domain ends at position 159, where the putative TM domain starts (up to residue 189). The CY domain is 375 residues long. The membrane-proximal region between amino acids 199 and 243 was deleted in the mutant TβRII Δ(200–242). B, human TβRI (ALK5; for sequence, see Ref. 8) consists of 503 amino acids. The last residue of the EC domain is at position 126, and the putative TM domain ends at residue 146. The sequence of the membrane-proximal region present in whole or in part in the TβRI truncation mutants is shown.

The membrane-proximal cytoplasmic region (amino acids 200–220) is involved in TβRII homomeric complex formation. COS7 cells were co-transfected by pairs of vectors encoding HA- and myc-tagged versions of the same TβRII construct (Fig. 1A). In control experiments, myc-TβRII was replaced by myc-tagged IL-9 receptor α subunit (myc-IL-9Rα) or Japan HA-2A520. At 44–48 h post-transfection, live cells were labeled consecutively in the cold (to eliminate endocytosis) by a series of antibodies to mediate patching and fluorescent labeling (see “Experimental Procedures”). In control experiments with Japan HA-2A520, the primary α-myc IgG was replaced by Fc125 mouse α-Japan HA. In experiments with ligand (250 pm TGF-β1), it was added at 4 °C 30 min before labeling with IgGs and maintained during subsequent incubations. The patching protocol results in HA-tagged receptors labeled by Alexa 594 (red) and myc-tagged receptors by Alexa 488 (green). Bars, 10 μm(A) and 2 μm(C). A, a typical image of a cell transfected with wt myc-TβRII and wt HA-TβRII and subjected to co-patching in the absence of ligand. White borders depict regions of interest (10 × 10 μm) analyzed for co-patching and for randomized co-localization (panels B and C, respectively). B, a zoomed image of region I in panel A. Using an algorithm written for Image-Pro Plus, green and red patches were defined as overlapping if their centers were separated by less than 0.2 μm (39). C, a zoomed randomized image to evaluate random overlap. The image was generated by merging the red channel of region I with the unrelated green channel of region II. D, computer analysis results of the specific images shown in panels B and C. E, averaged data (20–35 cells in each case) quantifying homomeric complex formation between TβRII constructs. The numbers of red (R), green (G), and overlapping (yellow, Y) patches were determined by computerized analysis as described (39), and the randomized control value was subtracted. The % co-patching (% of one tagged receptor in mutual patches with receptor carrying the other tag) is given by 100 × (Y/(Y + R)) for the red-labeled receptors and by 100 × (Y/(Y + G)) for the green-labeled receptors. Because these values were very close, a single mean ± S.E. value is depicted for each receptor pair. The homomeric co-patching of wt TβRII (∼25%) was mildly but significantly increased upon the addition of TGF-β1(p < 10^-4, Student's t test) and so was the case for all mutants except Ser-199 and Δ(200–242) (p > 0.05). Comparison of the % co-patching of each mutant without and with ligand to the levels measured similarly for wt TβRII shows a highly significant reduction for Ser-199 and Δ(200–242); ^**, p < 10^-6. Mutation of the single TβRII endocytosis signal (TβRII-3A) had only a minor effect on the co-patching in the absence of ligand (^*, p < 0.05) and no significant effect in the presence of ligand (p > 0.4).

Immunofluorescence co-patching demonstrates that TβRI homomeric complex formation does not require the cytoplasmic domain. COS7 cells were co-transfected with pairs of HA-TβRI and myc-TβRI or their truncation mutants. Control experiments employed co-transfection by a vector encoding wt HA- or myc-tagged TβRI together with either an unrelated raft-resident protein (X:31 BHA-PI) or an unrelated non-raft HA mutant (Japan HA-2A520), which were then patched using rabbit α-X:31 HA or mouse α-Japan HA as primary antibodies. Immunofluorescence co-patching experiments were performed and analyzed as detailed in Fig. 2. Bars are the mean ± S.E. of 20–40 cells in each case. The average level of co-patching in the randomized images (∼10%) was subtracted. TβRI displayed a high level of co-patching, suggesting extensive homo-oligomerization. Truncation of nearly all the CY domain (the Val-158 mutant) resulted in a significant further increase in co-patching as compared with full-length TβRI (^**, p < 10^-7).

Immunofluorescence co-patching reveals involvement of the CY domains of TβRII and TβRI in heteromeric complex formation. COS7 cells were co-transfected transiently with pairs of receptors carrying different epitope tags as indicated in panels A and B. Co-patching experiments were performed and analyzed as in Fig. 2. For experiments in the presence of ligand, TGF-β1 was added at 250 pm. Bars are the mean ± S.E. of experiments on 20–40 cells. A, heteromeric co-patching between wt myc-TβRI and HA-TβRII mutants. The co-patching measured for randomized images (∼10%) was subtracted. TGF-β1 mediated a highly significant increase (p < 10^-8) in the co-patching of TβRI with full-length TβRII and either wt, kinase-dead (K277R) or endocytosis-defective (the 3A mutant), and a significant TGF-β-mediated increase (p < 10^-4) was retained for TβRII truncations down to Pro-243 (the Val-419 and Pro-243 mutants). However, further truncations (at Leu-220 or Ser-199) or deletion of the 200–242 segment of TβRII resulted in a loss of the ligand-induced effect (p > 0.05). For each set of conditions (without and with ligand), the co-patching of wt myc-TβRI with each HA-TβRII mutant was compared with the basic co-patching level between the two wt receptors under similar conditions. Asterisks indicate significant differences from these basic levels (^***, p < 10^-13; ^**, p < 10^-8;^*, p < 10^-3). B, co-patching between wt HA-TβRII and myc-TβRI mutants. Randomized co-patching (∼10%) was subtracted. TGF-β1 strongly enhanced the co-patching between the wt receptors (p < 10^-7), and a lower but significant ligand effect was retained upon truncation of TβRI (p < 10^-3). The heteromeric co-patching of TβRII with kinase-dead TβRI (K232R mutation) was insensitive to the ligand (p > 0.05), although its ability to hetero-oligomerize with TβRII was not compromised. The co-patching of wt HA-TβRII with each myc-TβRI mutant was compared with its co-patching level with wt myc-TβRI under similar conditions (without or with ligand); asterisks indicate significant differences from these basic levels (^***, p < 10^-13; ^**, p < 10^-8).

Patch/FRAP studies demonstrate stable TβRII homomeric complexes which are lost in the Δ(200–242) mutant. COS7 cells were co-transfected with pairs of expression vectors encoding myc- and HA-tagged TβRII or Δ(200–242). In control experiments with singly expressed myc-tagged receptors, the HA-tagged construct was replaced by empty vector (left-most bars). 44–48 h after transfection live cells were subjected to the patching/labeling protocol described under “Experimental Procedures,” resulting in HA-tagged receptors patched and labeled by Cy3-GαR IgG, whereas the myc-tagged counterparts are labeled exclusively by Alexa 488-coupled non-cross-linking Fab′. FRAP experiments were conducted at 15 °C to avoid internalization. Where indicated, TGF-β1 (250 pm) was added at 4 °C 30 min before incubation with the antibodies and maintained at all the following stages. Controls experiments (no cross-linking) were conducted similarly, except that exclusive Fab′ labeling was employed. A, a representative FRAP curve of the lateral diffusion of myc-TβRII in a cell co-expressing HA-TβRI (no IgG cross-linking; control). B, a representative FRAP curve showing immobilization of IgG-cross-linked HA-TβRII. Only the R_F value is shown, as the recovery was too low to allow determination of D. C and E, average R_F values derived from multiple experiments. D and F, average D values derived from the patch/FRAP measurements. Bars are the mean ± S.E. of 30–40 measurements in each case. Asterisks indicate significant differences between the R_F values of the pairs indicated by brackets (^**, p < 10^-3; Student's t test). No significant differences were found in the D values as a result of IgG-mediated cross-linking, comparing each bar to the co-expressed uncross-linked control (p > 0.4).

Patch/FRAP studies demonstrate stable homomeric TβRI and TβRI-Val-158 complexes. COS7 cells were co-transfected with pairs of differently tagged (myc and HA) TβRI or TβRI-Val-158 (see “Experimental Procedures”). Patching/labeling and patch/FRAP experiments were performed without ligand, as TβRI does not bind ligand on its own. A and C, average R_F values. B and D, average D values. Bars are the mean ± S.E. of 30–40 measurements in each case. Asterisks indicate differences between the R_F values of the pairs indicated by brackets (^**, p < 10^-5; Student's t test). No significant differences were observed between the D values relative to the control lacking IgG cross-linking (p > 0.5).

Patch/FRAP studies demonstrate stable heteromeric interactions between TβRI and TβRII and distinguish between mutants based on the modulation of their interactions by TGF-β1. COS7 cells were co-transfected by myc-TβRI and an HA-tagged TβRII construct. Patching/labeling and patch/FRAP experiments were performed as in Fig. 5. Similar results (not shown) were obtained when the IgG/Fab′ labeling was swapped (IgG patching of myc and Fab′ labeling of the HA-tagged receptors). A, C, and E, average R_F values. B, D, and F, average D values. Bars are mean ± S.E. of 30–40 measurements in each case. Asterisks indicate significant differences between the R_F values of the pairs indicated by brackets (^*, p < 0.05; ^**, p < 10^-3; ^***, p < 10^-5).