Persistent cAMP signaling by thyrotropin (TSH) receptors is not dependent on internalization

Abstract

Evidence was presented that thyrotropin [thyroid-stimulating hormone (TSH)]-stimulated persistent cAMP signaling is dependent on receptor (with G-protein α subunits and adenylyl cyclase) internalization. Because it is not clear whether G proteins and adenylyl cyclase internalize with receptors, we tested whether persistent cAMP signaling by TSH receptor (TSHR) is dependent on internalization. We measured persistent TSHR signaling as an accumulation of cAMP in HEK-EM293 cells permanently expressing human TSHRs incubated with isobutylmethylxanthine for 30 min after washing the cells to remove unbound TSH, and TSHR internalization by fluorescence microscopy using Alexa-tagged TSH and binding assays using (125)I-TSH. TSHRs, but not the closely related lutropin or follitropin receptors, exhibit persistent cAMP signaling. TSHRs were not internalized by 30 min incubation with unlabeled TSH; however, expression of β-arrestin-2 promoted TSHR internalization that was inhibited by dynasore, a dynamin inhibitor. Expression of β-arrestin-2 had no effect on TSHR cAMP signaling, dynasore inhibited TSHR cAMP signaling in the absence or presence of TSHR internalization, and expression of a dominant-negative mutant dynamin, which inhibited internalization, had no effect on persistent cAMP signaling. Persistent cAMP signaling was specifically inhibited by a small molecule TSHR antagonist. We conclude that TSHRs do not have to be internalized to exhibit persistent cAMP signaling.

Figure 1.

Persistent cAMP signaling by TSHRs. A) Time course of acquisition of persistent signaling. HEKTSHR cells were incubated without (nothing) or with 10 mU/ml TSH at 37°C. At the times indicated, cells were washed 3 times with ice-cold acid buffer, followed by 1 wash with HBSS/HEPES, and then incubated in HBSS/HEPES with 1 mM IBMX or IBMX and 100 mU/ml TSH at 37°C for 30 min. Cells were lysed, and cAMP was measured in the cell lysates. Bars represent mean ± range of duplicate measurements in 1 of 2 experiments. B) Persistence of cAMP signaling. HEKTSHR cells were incubated without (nothing) or with 10 mU/ml TSH. After 30 min, cells were washed 3 times with ice-cold acid buffer, followed by 1 wash with HBSS/HEPES. Immediately after the washes, cells were incubated in HBSS/HEPES at 37°C. At 30, 60, 90, 120, and 180 min after the washes, cells were incubated in HBSS/HEPES with 1 mM IBMX or IBMX and 100 mU/ml TSH at 37°C for 30 min. Cells were lysed, and cAMP was measured in the cell lysates. Bars represent mean ± range of duplicate measurements in 1 of 4 experiments.

Figure 2.

Comparison of persistent cAMP signaling in cells stably expressing TSHRs, LHCGRs, or FSHRs. HEK-EM 293 cells stably expressing TSHRs, LHCGRs, or FSHRs were incubated without (nothing) or with 10 mU/ml TSH, 100 ng/ml LH, or 200 ng/ml FSH, respectively. After 30 min, cells were washed 3 times with ice-cold acid buffer, followed by 1 wash with HBSS/HEPES. At the times indicated (+min), cells were incubated in HBSS/HEPES with 1 mM IBMX or IBMX and 100 mU/ml TSH, 100 ng/ml LH, or 200 ng/ml FSH, respectively, at 37°C for 30 min. Cells were lysed, and cAMP was measured in cell lysates. Bars repreasent mean ± range of duplicate measurements in 2 experiments.

Figure 3.

βArrestin2 (βArr2) induces TSH-mediated TSHR internalization in HEKTSHR cells. A) TSH does not down-regulate TSHRs. HEKTSHR cells were incubated without (control) or with 100 mU/ml TSH. After 30 min, cell surface TSHRs were estimated by binding ¹²⁵I-TSH (6×10⁴ cpm) to cells in the cold for 3 h. Nonspecific binding was subtracted. Bars repressent mean ± range of duplicate measurements of specific binding in 2 experiments. AW, acid wash. B) βArr2 induces TSHR internalization stimulated by Alexa-TSH. HEKTSHR cells were transfected to transiently express GFP without or with βArr2 or with K44A. After 48 h, cells were incubated with Alexa-TSH for 30 min at 37°C. Images were acquired as described in Materials and Methods. Micrographs are representative of slices at the midpoint of the cell thickness. C) βArr2 increases acid-resistant binding to TSHRs. HEKTSHR cells were transfected with empty plasmid (mock) or with plasmid to express βArr2. After 48 h, the cells were incubated with ¹²⁵I-TSH at 37°C for 30 min. The cells were washed 3 times with ice-cold HBSS (total binding) or ice-cold acid buffer (acid-resistant binding) and the cell radioactivity counted. Percentage acid-resistant binding was calculated as 100 × (specific binding after acid wash/specific binding after HBSS wash). Bars represent mean ± sd of triplicate determinations in 2 experiments.

Figure 4.

βArrestin2 (βArr2) has no effect on persistent cAMP signaling. HEKTSHR cells were transfected with empty plasmid (mock) or with plasmid to express βArr2. After 48 h, the cells were incubated without (nothing) or with 100 mU/ml TSH at 37°C. After 30 min, the cells were washed 3 times with ice-cold acid buffer, followed by 1 wash with HBSS/HEPES. Immediately after the washes, cells were incubated in HBSS/HEPES at 37°C. At 120 min after the washes, cells were incubated in HBSS/HEPES with 1 mM IBMX or IBMX and 100 mU/ml TSH at 37°C for 30 min. Cells were lysed, and cAMP was measured in cell lysates. Bars represent mean ± range of duplicate measurements in 1 of 3 experiments.

Figure 5.

Dynasore nonspecifically inhibits TSHR persistent cAMP signaling. A) Dynasore inhibits TSHR internalization in βArr2-expressing HEKTSHR cells. HEKTSHR cells were transfected with empty plasmid (mock) or with plasmid to express βArr2. After 48 h, cells were incubated without (control) or with 200 or 400 μM dynasore in binding buffer. After 30 min, cells were incubated in binding buffer with dynasore and ¹²⁵I-TSH at 37°C for 30 min. Cells were washed 3 times with ice-cold HBSS (total binding) or ice-cold acid buffer (acid-resistant binding), and cell radioactivity was counted. Percentage acid-resistant binding was calculated as 100 × (specific binding after acid wash/specific binding after HBSS wash). Bars represent mean ± range of duplicate determinations in one of 2 experiments. B) Dynasore inhibits persistent TSHR cAMP signaling in a manner independent of internalization. HEKTSHR cells were transfected with empty plasmid (mock) or with plasmid to express βArr2. After 48 h, cells were incubated without (control) or with 400 μM dynasore in binding buffer. After 30 min, cells were incubated in binding buffer with dynasore and 100 mU/ml TSH at 37°C. After an additional 30 min, cells were incubated in HBSS/HEPES for 2 h and then in HBSS/HEPES with 1 mM IBMX or IBMX and 100 mU/ml TSH at 37°C for 30 min. Cells were lysed, and cAMP was measured in cell lysates. Bars represent mean ± range of duplicate measurements in 3 experiments.

Figure 6.

A dominant negative dynamin (K44A) inhibits βArr2-induced internalization but has no effect on persistent cAMP signaling by TSHR. A) Effect of K44A on acid-resistant ¹²⁵I-TSH binding. HEKTSHR cells were transfected with empty plasmid (mock), plasmid to express K44A, plasmid to express βArr2, or plasmids to coexpress K44A and βArr2. After 48 h, cells were incubated in binding buffer with ¹²⁵I-TSH at 37°C for 30 min. Cells were washed 3 times with ice-cold HBSS (total binding) or ice-cold acid buffer (acid-resistant binding), and cell radioactivity was counted. Percentage acid-resistant binding was calculated as 100 × (specific binding after acid wash/specific binding after HBSS wash). Bars represent mean ± range of duplicate determinations in 1 of 2 experiments. B) K44A has no effect on persistent cAMP signaling by TSHR. HEKTSHR cells were transfected with empty plasmid (mock), plasmid to express K44A, plasmid to express βArr2, or plasmids to coexpress K44A and βArr2. After 72 h, cells were incubated in HBSS/HEPES with 100 mU/ml TSH at 37°C. After 30 min, cells were incubated in HBSS/HEPES for 2 h and then in HBSS/HEPES with 1 mM IBMX or IBMX and 100 mU/ml TSH at 37°C for 30 min. Cells were lysed, and cAMP was measured in cell lysates. Bars represent mean ± se of duplicate measurements in 3 experiments.

Figure 7.

A small molecule TSHR antagonist added 2 h after TSH was removed inhibits persistent cAMP signaling by TSHR. HEKTSHR cells were incubated with 100 mU/ml TSH at 37°C. After 30 min, cells were washed 3 times with ice-cold acid buffer, followed by 1 wash with HBSS/HEPES. Immediately after the washes, cells were incubated in HBSS/HEPES at 37°C. After an additional 120 min, cells were incubated in HBSS/HEPES with 1 mM IBMX; IBMX and 100 mU/ml TSH; IBMX and 30 μM S2; or IBMX, S2, and TSH at 37°C for 30 min. Cells were lysed, and cAMP was measured in cell lysates. Bars represent mean ± range of duplicate measurements in 1 of 4 experiments.