Distinct endocytic responses of heteromeric and homomeric transforming growth factor beta receptors

Abstract

Transforming growth factor beta (TGF beta) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGF beta receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGF beta receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF beta receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGF beta receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGF beta receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGF beta receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGF beta receptors and that TGF beta receptor heteromers and homomers show distinct trafficking behavior.

Figure 1

Internalization of chimeric receptors. Individual clones were cultured as described in MATERIALS AND METHODS and incubated with 100 pM radiolabeled GM-CSF for 2 h at 4°C. Unbound ligand was removed, and cells were placed at 37°C for the indicated times. Surface-bound ligand was stripped by washes with PBS, pH 3.0, and radioactivity was measured. The remaining cell pellet was solublized and measured as internalized ligand. Each curve for each clone represents the mean ± SEM of two (A105), three (A122), or four (A120 or A110) independent experiments assayed in duplicate. The specific endocytotic rate constant (k_e) was determined for each of the clones over the first four data points (A105, 0.01/min; A110, 0.02/min; A120, 0.02/min; A122, 0.01/min). Analysis of variance shows no statistical difference between the rates (p > 0.60). The initial and maximal percent internalization, respectively, for clone A105 was 10.6% and 53.0%, for clone A110 was 7.5% and 51.9%, for clone A120 was 6.3% and 42.9%, and for clone A122 was 5.3% and 43.0%.

Figure 2

Clathrin-dependent internalization. (A) Heteromeric receptor expressing clone A105 was K⁺ depleted (▪) or placed in the identical buffers supplemented with 10 mM KCl (□) as described in MATERIALS AND METHODS. Internalization of ¹²⁵I-labeled GM-CSF was determined at the indicated times. (B) Clone A105 was treated with (▪) or without (□) NH₄Cl and amiloride at 37°C as described in MATERIALS AND METHODS. Cells then underwent 100 pM radiolabeled GM-CSF binding with or without amiloride for 2 h at 4°C and internalization proceeded as described in Figure 1. (Inset) AKR-2B cells were treated with (open bars) or without (solid bars) NH₄Cl and amiloride. After treatment, 150 pM EGF was bound at 4°C for 2 h. Internalization was initiated by removing unbound ligand and warming to 37°C for 0, 5, or 10 min. (C and D) Homomeric clones A120 (type I/type I; C) and A122 (type II/type II; D) were K⁺ depleted (▪) or supplemented with 10 mM KCl (□) as discussed in A and MATERIALS AND METHODS. Cells bound 100 pM radiolabeled GM-CSF in appropriate buffer B for 2 h at 4°C and internalization was determined at the times indicated. The results represent the mean ± SEM of two independent experiments done in duplicate.

Figure 3

Effect of potassium depletion on HRP accumulation. Control (□) or potassium-depleted (•) A105 cells were incubated on ice for 15 min with 2 mg/ml HRP. The cultures were then placed at 37°C and at the indicated times internalized HRP was determined and normalized to total cellular protein. The data represent the mean ± SEM of three separate experiments done in triplicate and the R² value was 0.95 and 0.87 for control and potassium-depleted cultures, respectively.

Figure 4

Down-regulation of chimeric receptor surface binding. Individual clones A120αIβI (•), A122αIIβII (○), A105αIβII (▪), and A110αIIβI (□) were treated with 10 ng/ml (520 pM) GM-CSF for the indicated times. Cells were acid stripped, and specific surface binding of 100 pM radiolabeled GM-CSF was determined after a 2-h incubation at 4°C with or without a 25-fold excess of unlabeled GM-CSF. Percentage of control binding is calculated as the percent of zero time (no prior GM-CSF treatment) specific binding. Each curve represents the mean ± SEM of three independent experiments assayed in duplicate.

Figure 5

Dose and temperature requirements for heteromeric receptor down-regulation. (A) Chimeric receptor down-regulation in clone A105 was induced by treatment with the indicated amounts of GM-CSF at 37°C for 2 h. After incubation, cells were acid stripped of ligand and remaining receptor surface binding was determined as described in Figure 4. The data represent the mean ± SEM of two independent experiments performed in duplicate. (B) Cells were treated with 10 ng/ml GM-CSF for the indicated times at either 4°C (▪), 25°C (□), or 37°C (•). Acid stripping and receptor surface binding were performed after each time point. Data are reported as the percent of binding compared with zero time for each dose or temperature. The data represent the mean ± SEM of three independent experiments performed in duplicate.

Figure 6

Recovery from down-regulation of surface binding. Heteromeric clone A105 was treated with 10 ng/ml GM-CSF for 4 h at 37°C. Cells were acid stripped and returned to 37°C in DMEM supplemented with 5% FBS (□), 5% FBS plus 1.0 μg/ml actinomycin D (•), or 5% FBS plus 20.0 μg/ml cycloheximide (○). At the indicated times, specific ¹²⁵I-labeled GM-CSF binding (100 pM) was determined after a 2-h incubation at 4°C. Percent binding of control is the ratio of specific binding observed after each treatment time to specific binding found in cells initially down-regulated. The data represent the mean ± SEM of three or four independent experiments done in duplicate. Analysis of variance between the 8-h FBS alone and 8-h FBS plus actinomycin D points showed no statistical difference (p = 0.13).