The Human Thyrotropin Receptor Activates G-proteins

The human thyrotropin receptor leads upon activa- tion to the stimulation of phospholipase C and adenylyl cyclase. It is presently not known whether this bifurcat-ing signaling occurs via two different G-proteins (Gq/,, and GB) or via one G-protein (GJ Receptor-activated G, releases P-y subunits and as, which then could regulate phospholipase C and adenylyl cyclase, respectively. In order to elucidate the signaling pathways induced by the activated thyroid-stimulating hormone (TSH) receptor, we studied the coupling of the TSH receptor to G, and Gdll in human thyroid membranes. TSH concentration dependently led to the activation of two forms of G, (G, short and G, as well as of G, and G,,, demonstrating that signaling pathways induced by TSH already bifurcate in the course of the receptor-<=- protein interaction. These data strongly suggest the con-cept that phospholipase C and adenylyl cyclase activation through the TSH receptor are mediated by GdI1 and G,, respectively.

tion through the TSH receptor are mediated by GdI1 and G,, respectively. Thyrotropin or thyroid-stimulating hormone (TSH)' is the main physiological agent implicated in the regulation of the thyroid gland. TSH controls the metabolism of the thyroid by activating a receptor that belongs to the family of G-proteincoupled receptors (1). Studies performed on human thyroid slices have shown that this receptor is able to stimulate two different regulation cascades: the adenylyl cyclase pathway and the hydrolysis of phosphatidylinositol bisphosphate by phospholipase C (2)(3)(4). Adenylyl cyclase is stimulated at lower TSH concentrations (0.1-1 milliunitslml) than phospholipase C (1-10 milliunitdml). This dual stimulation of the cascades is * This work was supported by the Ministere de la Politique Scientifique (P.A.I.), the Fonds National de la Recherche Scientifique (F.N.R.S.-TELEVIE), the European Economic Community Biomed program, and the Deutsche Forschungsgemeinschaft. 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  Dual signaling elicited by a single receptor has been shown for other receptors. At least three types of dual coupling have been observed among the receptors coupled to G-proteins that modulate adenylyl cyclase; in some, the receptors inhibit adenylyl cyclase and stimulate phospholipase C (5-7); in others as for the TSH receptor, they stimulate both adenylyl cyclase and phospholipase C (8-111, and in some, at the same time they activate the adenylyl cyclase system and inhibit its stimulation (12,13). In the first two examples of dual coupling, phospholipase C stimulation often requires higher concentrations of ligand than adenylyl cyclase modulation. However, the inverse relation has also been demonstrated for tachykinin receptors (14).
Recently, several groups have shown that G-protein Py dimers released upon activation of certain G-proteins by activated receptors are able, like the a subunits of G-proteins of the G, family, to activate p-isoforms of phospholipase C (15-17). Thus, phospholipase C stimulation by receptors stimulating or inhibiting adenylyl cyclase can be interpreted either in terms of coupling to two different G proteins (G, and a member of the G, class, or Gi and a member of the G, class) or in terms of coupling only to G, or G, and subsequent generation of G,-or G,-derived Py dimers (18). In this report, we demonstrate that the human TSH receptor activates both G, and Gdll, strongly suggesting that the TSH receptor activates adenylyl cyclase and phospholipase C via different G-proteins.

MATERIALS AND METHODS
Membrane Preparation-Human thyroid membranes were prepared from normal gland (partly removed in the resection of laryngeal cancer) or from paranodular tissue obtained from patients undergoing thyroidectomy for solitary nodules. The tissues were frozen in liquid N, soon after dissection. For membrane preparation, the tissues were ground into a fine powder under liquid N, and then homogenized in a glass-Teflon homogenizer in 15 m M Tris-HC1 (pH 7.5) containing 2 m~ MgCl,, 0.3 m M EDTA, 1 m~ EGTA, 0.3 m M phenylmethylsulfonyl fluoride, and 5 p~ leupeptin. The homogenate was filtered through two layers of gauze. After a low speed centrifugation (10 min at 120 x g ) the supernatant was carefully removed and subjected to a 30-min centrifugation at 40,000 x g. The final pellet was resuspended in 50 m~ Tris-HC1 (pH 7.5) containing 10 m M MgSO, and 0.5 m~ EDTA. A protein measurement using the method of Larson (19) was performed, and the membrane suspension was stored at -80 "C.
Photolabeling of Membrane Protein~-[n-~~P]GTP azidoanilide was synthesized and purified as described (20). Cell membranes were suspended in an ice-cold incubation buffer containing 100 m~ Hepes pH 7.4, 0. Sample tubes with 30 pl of the membrane suspension were preincubated for 3 min at 30 "C with 10 pl of TSH (different concentrations, 0-1000 milliunits/ml). Thereafter, 20 pl of [cx-~'P]GTP azidoanilide, diluted in water, were added (2 pCi/tube; specific activity, 3000 Ci/mmol), and after 10 min, the reaction was stopped by putting the samples on ice. All the subsequent procedures were performed at 4 "C. The samples were centrifuged (12,000 x g for 5 rnin), and membrane pellets were resuspended in 60 pl of the photolysis buffer containing 50 m~ Hepes pH 7.4, 0.1 m M EDTA, 10 m M MgCl,, 30 m M NaCl, 1 m M benzamidine, and 2 m~ glutathione. The samples were then irradiated for 15 s at 4 "C with an ultraviolet lamp (254 nm, 150 watts) from a distance of 3 cm. After irradiation, samples were again centrifuged (12,000 x g for 5 rnin).

13733
SDS-PAGE, Autoradiography, and Immunoblotting-SDS-PAGE was performed on 13% (w1v) acrylamide gels, and gels were run until the 30-kDa standard protein reached the bottom of the gel (23). Photolabeled proteins were visualized by autoradiography of dried gels with Kodak X-OMAT AR-5 films. Blotting of membrane proteins separated by SDS-PAGE, processing of immunoblots, and detection of immunoreactive proteins by a chemiluminescence procedure have been described (24). Antisera raised against peptides corresponding to specific regions of G-protein a subunits are shown in Table I. Specificity of antisera was controlled by testing their selectivity for a subunits expressed in Escherichia coli.

RESULTS
In order to determine which G-proteins are expressed in human thyroid cells, Western blots have been performed. Six different antibodies raised against peptides specific for individual G-protein a subunits have been used: AS 370, anti-a,,,,; AS 348, anti-a,; AS 266, anti-a,; AS 233, anti-a,,; AS 343, anti-aI3; and AS 255, anti-a,, (see Table I). Western blots show clearly that the human thyroid contains two forms of G,, with a subunits of 45 kDa (asshort) and 52 kDa (aslong) as well as G, and G,, with a subunits exhibiting molecular masses of 42 and 43 kDa, respectively (Fig. 1). In addition, we found Gi-type G-proteins, GI, and GIs, with a subunits migrating at molecular masses of 40, 44, and 42 kDa, respectively (Fig. 1).
To study the coupling of the activated TSH receptor to G,,, and G,, human thyroid membranes were photolabeled with [C~-~'P]GTP azidoanilide in the presence of various concentrations of TSH (0-1000 milliunits/ml). Thereafter, samples were split, the photolabeled a subunits were immunoprecipitated with the antiserum (AS 370) or with the a, antiserum (AS 348) (Fig. 2, A and B, respectively), and immunoprecipitated proteins were separated by SDS-PAGE. Immunoprecipitation with the aq,,, antiserum revealed the incorporation of [a-"P1GTP azidoanilide into the 42-and 43-kDa a subunits of G,,,, in the presence of increasing TSH concentrations. The 43-kDa protein corresponds to the a,, subunit whereas the 42-kDa protein represents the aq subunit (see Fig. 1). Using the same SDS-gel system, Berstein et al. (25) have found identical migration properties for aq and a,,. Fig. 2B shows the agonistdependent stimulation of photolabeling of the two forms of G,  255 (1/150); lane 3, a,, AS 348 (11150); lune 4, a,,, AS 343 (11150); lune 5, aI2, AS 233 (11150); lune 6, ai, AS 266 (1112.5). with a subunits of 45 and 52 kDa. Preimmune antisera did not precipitate any photolabeled proteins, demonstrating that precipitation with the employed antisera was specific. Thus, incubation of thyroid membranes with TSH induced the activation of G,,,, and both forms of G, (Gsshort and GSlong). Significant activation of the G-proteins (G, short and G, as well as G, and GI,) by TSH was observed at the same concentration of TSH (10 milliunits/ml). Above 100 milliunits/ml TSH, the intensity of the stimulation seems to plateau. These concentrations of TSH are of the same order as those used for adenylyl cyclase activation in thyroid membranes (261, but they are higher than those necessary to activate cyclic AMP accumulation in intact cells (27). DISCUSSION Several receptors including the TSH receptor mediate activation of both adenylyl cyclase and phospholipase C . The mechanism by which this dual coupling occurs is presently not clear. Two hypotheses may explain how one receptor is able to activate the two effectors. One is that the receptor couples to two different G proteins (G, and a member of the G, family), and a subunits then regulate the activity of adenylyl cyclase and phospholipase C (Fig. 3). Another model implies that the &dimers freed upon G, activation stimulate the phospholipase C whereas the G, a subunits activate adenylyl cyclase (Fig. 3).
In the present study, we used the TSH receptor as a model system and studied its coupling to G, and Gq,,,. Our results clearly support the first hypothesis (Fig. 3, model 1 ) . Indeed, we demonstrated in human thyroid membranes the presence of both forms of as, a, and a,,, and showed by photolabeling of G-protein a subunits activated by the TSH receptor that this receptor couples to G, and G,,,,. This strongly suggests that TSH-induced activation of adenylyl cyclase and phospholipase C is mediated by G, and G,,,,, respectively. That TSH does not stimulate phospholipase C through Gi was already demonstrated by the fact that the TSH stimulation of the inositol bisphosphate cascade in human thyroid cells is not inhibited by pertussis toxin (not shown). Thus, in our system, a role for & dimers in the process of phospholipase C stimulation by the TSH receptor does not need to be suggested. As other extracellular signals such as luteinizing hormone, parathyroid hormone, and thyroliberin (8,9,11) have been shown to activate both adenylyl cyclase and phospholipase C it would be of great interest to know if the dual G-protein activation model also holds up for them.
In human thyroid slices as well as in Chinese hamster ovary cells transfected with the human TSH receptor, it has been shown that the stimulation of phospholipase C by the TSH receptor occurs at higher concentrations of ligand than the activation of adenylyl cyclase. A similar ligand concentrationeffect relationship has been shown for other receptors coupling to phospholipase C and adenylyl cyclase (5, 6, 9). Two hypotheses may account for the observed lower sensitivity of the phospholipase C pathway versus the adenylyl cyclase system. One would be that the affinity of the activated receptor is higher for G,-proteins than for G,-proteins involved in the respective pathways. Another would be that a difference exists between the efficacy of coupling of the G-proteins and their respective effectors. The photolabeling experiments might seem to support the second model, since no obvious difference in the potency of TSH to activate G, and G,,,, could be observed.
ling to Gs and G,,,,

13735
Interestingly, TSH concurrently led to the activation of G, and G,, as well as of both forms of G,. I t has been shown in several reports that both aq and a,, on one hand and a, short and a, on the other hand behave very similarly if not identically with regard to their downstream enzymatic effectors. Both a, and a,, have been shown to stimulate phospholipase C-p activity (28"30), and both forms of G, activate adenylyl cyclase (31, 32). Thus, it is tempting to speculate that some functional redundancy exists among the G,,,, as well as the G, forms of G-proteins.