甲状腺髓样癌细胞PTHR_1表达和GRK介导的AC-cAMP-PKA及Ca~(2+)/PKC信号传导通路研究
|
摘要
研究背景：甲状腺髓样癌来源于分泌降钙素的甲状腺滤泡旁细胞(c细胞),约占全部甲状腺癌的5-10%,其恶性程度介于分化性甲状腺癌与甲状腺未分化癌之间,预后相对较差,其死亡率约占所有甲状腺癌相关死亡病例的13.4%。由于c细胞不表达促甲状腺激素受体且不摄碘,传统的促甲状腺激素抑制疗法和131|核素内放射治疗无效,全身性化疗和放射治疗疗效欠佳,根治性外科手术成为绝大多数甲状腺髓样癌患者的唯一选择,治疗手段的匮乏和单一直接影响和制约甲状腺髓样癌患者的疗效和预后。关于甲状腺髓样癌发病过程中c细胞表面受体分布及内分泌激素的调控机制仍不清楚,而作为G蛋白偶联受体之一的甲状旁腺素受体,其在c细胞表面的表达、内化及调控及其信号通路与甲状腺髓样癌发病调控机制中的交互作用仍未见报道。
目的：明确甲状腺C细胞表面受体分布及PTHR1在甲状腺髓样癌组织中的表达,探索PTHR1信号通路和GRK介导的AC-cAMP-PKA信号系统和Ca2+/PKC信号系统信号通路在甲状腺髓样癌发病调控机制中的交互作用,为甲状腺髓样癌的治疗提供理论支撑与新的靶点。
方法：1.采用免疫组织化学法、实时荧光定量聚合酶链反应和蛋白免疫印迹法(Western blot)检测PTHR1, CTR、GRK2、GRK5、CaM、PKC及PKA在甲状腺髓样癌组织中的表达情况,分别从组织、蛋白和核酸水平深入研究GPCRs、 GRK表达与AC-cAMP-PKA信号系统和Ca2+/PKC信号系统信号通路的影响。2.通过甲状腺髓样癌TT细胞株的体外培养,经外源性干预添加PTH1-34和PTHR McAb,采用实时荧光定量聚合酶链反应和蛋白免疫印迹法(Western blot)检测PTHR1, CTR、GRK2、GRK5及CaM在甲状腺髓样癌TT细胞加药前后的表达情况,采用电化学发光法检测甲状腺髓样癌TT细胞株加药前后细胞培养上清液PTH和CT水平,采用放射免疫竞争结合法检测细胞内cAMP浓度和液闪记数γ-32P放射活性法检测PKC、PKC活性变化并探讨添加PTH1-34对于甲状腺髓样癌TT细胞的影响以及PTHR1表达与AC-cAMP-PKA信号系统和Ca2+/PKC信号系统信号通路。
结果：1.免疫组织化学法显示PTHR1、CTR微弱-中等表达于正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌组织并强表达于甲状腺髓样癌组织,表明该受体广泛分布于正常甲状腺滤泡上皮、甲状腺乳头状癌细胞及甲状腺C细胞,并以甲状腺髓样癌组织表达最为显著。其中,各组织类型组甲状腺肿瘤PTHR中等表达无差异性(p>0.05),而各组呈强阳性表达和弱阳性表达之间有显著性差异(p<0.05)；GRK2、GRK5表达于正常甲状腺、结节性甲状腺肿,二组表达之间无差异性(p>0.05)；GRK2在甲状腺乳头状癌呈强阳性表达而在甲状腺髓样癌组织呈弱阳性表达(p<0.05),GRK5在甲状腺乳头状癌和甲状腺髓样癌组织呈弱阳性表达。二组表达之间差异无显著性(p>0.05)。实时荧光定量聚合酶链反应结果显示在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间PTHR1mRNA、CTR mRNA表达无显著性差异(p>0.05)；与正常甲状腺组比较,甲状腺髓样癌组PTHR1mRNA, CTR mRNA表达有显著性差异(p<0.05)。在正常甲状腺组与结节性甲状腺肿组之间GRK2mRNA, GRK5mRNA表达无显著性差异(p>0.05)。甲状腺乳头状癌组GRK2mRNA和GRK5mRNA表达水平明显高于正常甲状腺组,二者表达有显著性差异(p<0.05)；而对于甲状腺髓样癌组,其GRK2mRNA表达水平明显高于正常甲状腺组而GRK5mRNA却明显低于正常甲状腺组,二者表达有显著性差异(p<0.05)。在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间PKA mRNA、PKC mRNA表达无显著性差异(p>0.05)；甲状腺髓样癌组PKA mRNA、PKC mRNA表达水平明显高于正常甲状腺组,二者表达有显著性差异(p<0.05)。其中,在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之CaM mRNA表达无显著性差异(p>0.05)；甲状腺髓样癌组CaM mRNA表达水平明显高于王常甲状腺组,二者表达有显著性差异(p<0.05)。
Western blot结果显示：在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间PTHR1、CTR蛋白表达无显著性差异(p>0.05)；与之比较,甲状腺髓样癌组PTHR1、CTR蛋白表达明显高于其余组,表达之间有显著性差异(p<0.05)。其中,在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间GRK2、GRK5蛋白表达无显著性差异(p>0.05)；与之比较,甲状腺髓样癌组GRK2蛋白表达明显高于其余组,而GRK5蛋白表达明显低于其余组,二者表达之间有显著性差异(p<0.05)。其中,在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间PKC、PKA蛋白表达无显著性差异(p>0.05)；与之比较,甲状腺髓样癌组PKC、PKA蛋白表达明显高于其余组,表达之间有显著性差异(p<0.05)。其中,在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间CaM蛋白表达无显著性差异(p>0.05)；与之比较,甲状腺髓样癌组CaM蛋白表达明显高于其余组,表达之间有显著性差异(p<0.05)。
2.F-12K培养基培养甲状腺髓样癌TT细胞,细胞呈长梭形、多边形或不规则形,贴壁生长,48-72小时后约80%TT细胞贴壁融合。给予PTH1-34作用后细胞数量明显减少,部分细胞出现胞体收缩变圆,细胞间隙进一步增宽,部分细胞呈现生长延缓,悬浮细胞及碎片增多；而给予PTHR McAb作用后细胞数量未见明显减少,部分细胞呈现生长延缓,细胞形态及活力未见明显改变。添加PTH1-34组TT细胞上清液上清液PTH水平明显升高p<0.05)而CT水平明显降低p<0.05),而不同剂量PTH1-34组TT细胞上清液水平之间无明显差异(p>0.05)。与甲状腺髓样癌TT细胞对照组相比较,加PTH1-34组TT细胞cAMP水平明显升高p<0.05),而PTHR McAb组TT细胞cAMP明显降低(p<0.05),不同剂量PTH1-34组TT细胞cAMP水平之间无明显差异(p>0.05)。实时荧光定量聚合酶链反应和蛋白免疫印迹法(Western blot)检测表明通过降低PKC活性、升高PKA活性,从而下调GRK2表达并上调GRK5表达,从而调节GPCRs表达。PTHR1蛋白表达明显升高而CTR蛋白表达明显降低,GRK2蛋白表达明显降低而GRK5蛋白表达均明显升高,同时CaM表达明显降低,组间比较有显著性差异(p<0.05)。
结论：
1.甲状腺C细胞膜表面表达PTHR1和CTR,同时,由于第二信使调节激酶(PKA、PKC)在甲状腺髓样癌组织中的高表达及CaM的高表达,导致G蛋白偶联受体激酶(GRK2、GRK5)的不同表达水平即GRK2的活性增强而GRK5的活性减弱,由于GRK2和GRK5对甲状腺C细胞膜表面GPCRs即PTHR1和CTR的不同强度的磷酸化作用,最终显示甲状腺髓样癌组织中PTHR1和CTR的不同程度的上调性表达。
2.通过外源性添加PTH1-34和PTHR McAb观察PTH1-34在体外对甲状腺髓样癌TT细胞株生长的影响,细胞培养上清液表明其PTH水平明显上升而CT水平下降；cAMP浓度升高同时PKA上升而PKC降低。同时CaM表达降低,GRK2表达降低而GRK5表达升高,最终激活AC-cAMP-PKA信号系统而抑制了Ca2+/PKC信号系统信号通路传导,导致甲状腺髓样癌TT细胞生长抑制,研究进一步揭示了PTHR1信号通路传导对恶性肿瘤细胞生长的影响,本研究提示PTHR1可能成为甲状腺髓样癌靶向治疗的靶点之一,为甲状腺髓样癌的治疗开辟了崭新的视角与思路。
Background:Medullary thyroid carcinoma originated from thyroid parafollicular cell (C cells) that secreted calcitonins. It accounts for about5-10%of all of thyroid cancer. The degree of malignancy between the differentiated thyroid carcinoma and anaplastic thyroid carcinoma, the prognosis was relatively poor, the mortality rate of MTC accounts for about13.4%of all thyroid cancer-related deaths. Due to C cells do not express the TSH receptor without uptake iodine so that the traditional thyroid-stimulating hormone suppressive therapy and131I radionuclide therapy were ineffective for MTC. Meanwhile it showed poor effect to systemic chemotherapy and radiotherapy, the radical surgery has become the unique option for the most patients with MTC. The efficacy and prognosis of the patients was restricted by the single and lacked treatment. It remains unclear for the C cell surface receptor distribution and regulatory mechanism of endocrine hormones in MTC. As one of the G protein-coupled receptor, it has not yet been reported that parathyroid hormone receptors in C cell surface expression, receptors internalization and interaction in the regulation of its signaling pathway and the incidence of MTC regulatory mechanism.
Objectives:To clear the thyroid C cell surface receptor distribution and PTHR1expression in medullary thyroid carcinoma tissue and explore the PTHR1signaling pathway and GRK-mediated AC-cAMP-PKA signaling system and Ca2+/PKC signaling system signaling pathway in the regulation mechanism and interaction of MTC. The project was trying to provide theoretical support and a new target for the treatment of medullary thyroid carcinoma.
Methods:(1) Using immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction and protein immunoblot method to detect PTHR1, CTR and GRK2, GRK5, CaM, PKC, and PKA in MTC tissue expression, respectively. From the organization, protein and nucleic acid level to study the expression of GPCRs and GRK, AC-cAMP-PKA signaling system, Ca2+/PKC signaling system signaling pathway.(2) Medullary thyroid carcinoma TT cell cultured in vitro by exogenous interventions add of PTH1-34and PTHR McAb. The expression of PTHR1CTR, GRK2, GRK5and CaM in MTC TT cells were detected by the real-time fluorescence quantitative polymerase chain reaction and protein immunoblot method before and after dosing. The PTH and CT level of the cell culture supernatant were detected by electrochemical luminescence, the intracellular cAMP concentration was detected by radioimmunoassay competitive binding assay and the activity of PKC and PKC were detected by liquid scintillation counting γ-32P radioactivity assay during MTC TT cell cultured in F-12culture medium. We try to explore the effect of medullary thyroid carcinoma TT cells, PTHR1expression and the AC-cAMP-PKA and Ca2+/PKC signaling pathway added of PTH1-34.
Results:(1) The Immunohistochemical staining showed PTHR1, CTR weak-medium expressed in normal thyroid, nodular goiter and thyroid papillary carcinoma and medullary thyroid carcinoma tissue. There was no significant difference between different tissue type group of thyroid tumors on PTHR1medium expression (p>0.05) and there was significant difference between different tissue type group of thyroid tumors on PTHR1strong positive expression (p<0.05).Expression of GRK2, GRK5was no difference between the normal thyroid and nodular goiter groups (p>0.05). GRK2expression was strong positive in papillary thyroid carcinoma and was weakly positive in medullary thyroid carcinoma tissue (p<0.05), GRK5expression were both weakly positive expression in papillary thyroid carcinoma and medullary thyroid carcinoma tissue. There was no significant difference between the two groups (p>0.05).Real-time fluorescence quantitative polymerase chain reaction showed there was no significant difference in the expression of the PTHR1mRNA CTR mRNA between the normal thyroid, nodular goiter and papillary thyroid carcinoma (P>0.05) but there was significant difference in the expression of the PTHR1mRNA CTR mRNA between the normal thyroid and medullary thyroid carcinoma (p<0.05). The expression of GRK2mRNA and GRK5mRNA was no significant difference between normal thyroid group and nodular goiter group (p>0.05). The expression of GRK2mRNA and GRK5mRNA in papillary thyroid carcinoma was significantly higher than normal thyroid group (p<0.05). GRK2mRNA expression was significantly higher than normal thyroid group and GRK5. mRNA expression was significantly lower than normal thyroid group in medullary thyroid carcinoma group which both showed the significant difference (p<0.05).Between normal thyroid, nodular goiter, thyroid papillary carcinoma of PKA mRNA and PKC mRNA expression was no significant difference (p>0.05); compared with normal thyroid group, the expression of PKA mRNA of PKC mRNA were significantly higher (p<0.05).There was no significant difference of expression CaM mRNA between normal thyroid, nodular goiter, thyroid papillary carcinoma (p>0.05).The expression CaM mRNA was higher in medullary thyroid carcinoma than normal thyroid that there was significant difference between two groups (p<0.05). Western blot showed same results as real-time fluorescence quantitative polymerase chain reaction.(2) Medullary thyroid carcinoma TT cells culture in F-12K medium showed long spindle, polygon or irregular shape to stick wall growth. It showed adherent growth of about80%of the48-to72hours after TT cells adherent fusion, give PTH1-34role significantly reduce cell number and some cells the cell body shrinkage and round cell gap is further widened, some cells showed growth delay, suspension cells and debris increased; given PTHR McAb to the role of cell number did not significantly reduce the Some cells showed a growth delay, cell morphology and vitality was no significant change. Add PTH1-34TT cell supernatant supernatant PTH levels significantly higher (p <0.05) and CT levels were significantly lower (p<0.05) between the different doses of PTH1-34TT supernatant level no significant difference (p>0.05).
Conclusions:(1) Thyroid C cell surface express PTHR1and CTR. Due to the high expression of the second messenger-regulated kinase (PKA and PKC) and CaM in medullary thyroid carcinoma tissue, G protein-coupled receptor kinase-GRK2and GRK5expression differently that GRK2activity enhance and GRK5activity weakened. As different phosphorylation of GRK2and GRK5on the thyroid C cell surface GPCRs, the result showed increase expression of PTHR1and CTR in medullary thyroid carcinoma tissue.(2) Add through the exogenous PTH1-34and the PTHR McAb, observe PTH1-34in vitro effects of medullary thyroid carcinoma TT cell line, cell culture supernatant showed that PTH levels were significantly increased CT levels; of cAMP concentration increased at the same time PKA rise in the PKC lower. At the same time CaM decreased expression of GRK2reduced expression of GRK5expression was elevated, and ultimately activate the AC-cAMP-PKA signaling system and inhibition of Ca2+/PKC signaling system signaling pathway conduction, resulting in medullary thyroid carcinoma TT cell growth inhibition, the study further revealed the PTHR1signaling pathway conduction growth of malignant cells, this study suggests that the PTHR1could become one of the targets of targeted therapy of medullary thyroid carcinoma, and opens up new perspectives and ideas for the treatment of medullary thyroid carcinoma.
目的：明确甲状腺C细胞表面受体分布及PTHR1在甲状腺髓样癌组织中的表达,探索PTHR1信号通路和GRK介导的AC-cAMP-PKA信号系统和Ca2+/PKC信号系统信号通路在甲状腺髓样癌发病调控机制中的交互作用,为甲状腺髓样癌的治疗提供理论支撑与新的靶点。
方法：1.采用免疫组织化学法、实时荧光定量聚合酶链反应和蛋白免疫印迹法(Western blot)检测PTHR1, CTR、GRK2、GRK5、CaM、PKC及PKA在甲状腺髓样癌组织中的表达情况,分别从组织、蛋白和核酸水平深入研究GPCRs、 GRK表达与AC-cAMP-PKA信号系统和Ca2+/PKC信号系统信号通路的影响。2.通过甲状腺髓样癌TT细胞株的体外培养,经外源性干预添加PTH1-34和PTHR McAb,采用实时荧光定量聚合酶链反应和蛋白免疫印迹法(Western blot)检测PTHR1, CTR、GRK2、GRK5及CaM在甲状腺髓样癌TT细胞加药前后的表达情况,采用电化学发光法检测甲状腺髓样癌TT细胞株加药前后细胞培养上清液PTH和CT水平,采用放射免疫竞争结合法检测细胞内cAMP浓度和液闪记数γ-32P放射活性法检测PKC、PKC活性变化并探讨添加PTH1-34对于甲状腺髓样癌TT细胞的影响以及PTHR1表达与AC-cAMP-PKA信号系统和Ca2+/PKC信号系统信号通路。
结果：1.免疫组织化学法显示PTHR1、CTR微弱-中等表达于正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌组织并强表达于甲状腺髓样癌组织,表明该受体广泛分布于正常甲状腺滤泡上皮、甲状腺乳头状癌细胞及甲状腺C细胞,并以甲状腺髓样癌组织表达最为显著。其中,各组织类型组甲状腺肿瘤PTHR中等表达无差异性(p>0.05),而各组呈强阳性表达和弱阳性表达之间有显著性差异(p<0.05)；GRK2、GRK5表达于正常甲状腺、结节性甲状腺肿,二组表达之间无差异性(p>0.05)；GRK2在甲状腺乳头状癌呈强阳性表达而在甲状腺髓样癌组织呈弱阳性表达(p<0.05),GRK5在甲状腺乳头状癌和甲状腺髓样癌组织呈弱阳性表达。二组表达之间差异无显著性(p>0.05)。实时荧光定量聚合酶链反应结果显示在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间PTHR1mRNA、CTR mRNA表达无显著性差异(p>0.05)；与正常甲状腺组比较,甲状腺髓样癌组PTHR1mRNA, CTR mRNA表达有显著性差异(p<0.05)。在正常甲状腺组与结节性甲状腺肿组之间GRK2mRNA, GRK5mRNA表达无显著性差异(p>0.05)。甲状腺乳头状癌组GRK2mRNA和GRK5mRNA表达水平明显高于正常甲状腺组,二者表达有显著性差异(p<0.05)；而对于甲状腺髓样癌组,其GRK2mRNA表达水平明显高于正常甲状腺组而GRK5mRNA却明显低于正常甲状腺组,二者表达有显著性差异(p<0.05)。在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间PKA mRNA、PKC mRNA表达无显著性差异(p>0.05)；甲状腺髓样癌组PKA mRNA、PKC mRNA表达水平明显高于正常甲状腺组,二者表达有显著性差异(p<0.05)。其中,在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之CaM mRNA表达无显著性差异(p>0.05)；甲状腺髓样癌组CaM mRNA表达水平明显高于王常甲状腺组,二者表达有显著性差异(p<0.05)。
Western blot结果显示：在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间PTHR1、CTR蛋白表达无显著性差异(p>0.05)；与之比较,甲状腺髓样癌组PTHR1、CTR蛋白表达明显高于其余组,表达之间有显著性差异(p<0.05)。其中,在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间GRK2、GRK5蛋白表达无显著性差异(p>0.05)；与之比较,甲状腺髓样癌组GRK2蛋白表达明显高于其余组,而GRK5蛋白表达明显低于其余组,二者表达之间有显著性差异(p<0.05)。其中,在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间PKC、PKA蛋白表达无显著性差异(p>0.05)；与之比较,甲状腺髓样癌组PKC、PKA蛋白表达明显高于其余组,表达之间有显著性差异(p<0.05)。其中,在正常甲状腺、结节性甲状腺肿、甲状腺乳头状癌各组之间CaM蛋白表达无显著性差异(p>0.05)；与之比较,甲状腺髓样癌组CaM蛋白表达明显高于其余组,表达之间有显著性差异(p<0.05)。
2.F-12K培养基培养甲状腺髓样癌TT细胞,细胞呈长梭形、多边形或不规则形,贴壁生长,48-72小时后约80%TT细胞贴壁融合。给予PTH1-34作用后细胞数量明显减少,部分细胞出现胞体收缩变圆,细胞间隙进一步增宽,部分细胞呈现生长延缓,悬浮细胞及碎片增多；而给予PTHR McAb作用后细胞数量未见明显减少,部分细胞呈现生长延缓,细胞形态及活力未见明显改变。添加PTH1-34组TT细胞上清液上清液PTH水平明显升高p<0.05)而CT水平明显降低p<0.05),而不同剂量PTH1-34组TT细胞上清液水平之间无明显差异(p>0.05)。与甲状腺髓样癌TT细胞对照组相比较,加PTH1-34组TT细胞cAMP水平明显升高p<0.05),而PTHR McAb组TT细胞cAMP明显降低(p<0.05),不同剂量PTH1-34组TT细胞cAMP水平之间无明显差异(p>0.05)。实时荧光定量聚合酶链反应和蛋白免疫印迹法(Western blot)检测表明通过降低PKC活性、升高PKA活性,从而下调GRK2表达并上调GRK5表达,从而调节GPCRs表达。PTHR1蛋白表达明显升高而CTR蛋白表达明显降低,GRK2蛋白表达明显降低而GRK5蛋白表达均明显升高,同时CaM表达明显降低,组间比较有显著性差异(p<0.05)。
结论：
1.甲状腺C细胞膜表面表达PTHR1和CTR,同时,由于第二信使调节激酶(PKA、PKC)在甲状腺髓样癌组织中的高表达及CaM的高表达,导致G蛋白偶联受体激酶(GRK2、GRK5)的不同表达水平即GRK2的活性增强而GRK5的活性减弱,由于GRK2和GRK5对甲状腺C细胞膜表面GPCRs即PTHR1和CTR的不同强度的磷酸化作用,最终显示甲状腺髓样癌组织中PTHR1和CTR的不同程度的上调性表达。
2.通过外源性添加PTH1-34和PTHR McAb观察PTH1-34在体外对甲状腺髓样癌TT细胞株生长的影响,细胞培养上清液表明其PTH水平明显上升而CT水平下降；cAMP浓度升高同时PKA上升而PKC降低。同时CaM表达降低,GRK2表达降低而GRK5表达升高,最终激活AC-cAMP-PKA信号系统而抑制了Ca2+/PKC信号系统信号通路传导,导致甲状腺髓样癌TT细胞生长抑制,研究进一步揭示了PTHR1信号通路传导对恶性肿瘤细胞生长的影响,本研究提示PTHR1可能成为甲状腺髓样癌靶向治疗的靶点之一,为甲状腺髓样癌的治疗开辟了崭新的视角与思路。
Background:Medullary thyroid carcinoma originated from thyroid parafollicular cell (C cells) that secreted calcitonins. It accounts for about5-10%of all of thyroid cancer. The degree of malignancy between the differentiated thyroid carcinoma and anaplastic thyroid carcinoma, the prognosis was relatively poor, the mortality rate of MTC accounts for about13.4%of all thyroid cancer-related deaths. Due to C cells do not express the TSH receptor without uptake iodine so that the traditional thyroid-stimulating hormone suppressive therapy and131I radionuclide therapy were ineffective for MTC. Meanwhile it showed poor effect to systemic chemotherapy and radiotherapy, the radical surgery has become the unique option for the most patients with MTC. The efficacy and prognosis of the patients was restricted by the single and lacked treatment. It remains unclear for the C cell surface receptor distribution and regulatory mechanism of endocrine hormones in MTC. As one of the G protein-coupled receptor, it has not yet been reported that parathyroid hormone receptors in C cell surface expression, receptors internalization and interaction in the regulation of its signaling pathway and the incidence of MTC regulatory mechanism.
Objectives:To clear the thyroid C cell surface receptor distribution and PTHR1expression in medullary thyroid carcinoma tissue and explore the PTHR1signaling pathway and GRK-mediated AC-cAMP-PKA signaling system and Ca2+/PKC signaling system signaling pathway in the regulation mechanism and interaction of MTC. The project was trying to provide theoretical support and a new target for the treatment of medullary thyroid carcinoma.
Methods:(1) Using immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction and protein immunoblot method to detect PTHR1, CTR and GRK2, GRK5, CaM, PKC, and PKA in MTC tissue expression, respectively. From the organization, protein and nucleic acid level to study the expression of GPCRs and GRK, AC-cAMP-PKA signaling system, Ca2+/PKC signaling system signaling pathway.(2) Medullary thyroid carcinoma TT cell cultured in vitro by exogenous interventions add of PTH1-34and PTHR McAb. The expression of PTHR1CTR, GRK2, GRK5and CaM in MTC TT cells were detected by the real-time fluorescence quantitative polymerase chain reaction and protein immunoblot method before and after dosing. The PTH and CT level of the cell culture supernatant were detected by electrochemical luminescence, the intracellular cAMP concentration was detected by radioimmunoassay competitive binding assay and the activity of PKC and PKC were detected by liquid scintillation counting γ-32P radioactivity assay during MTC TT cell cultured in F-12culture medium. We try to explore the effect of medullary thyroid carcinoma TT cells, PTHR1expression and the AC-cAMP-PKA and Ca2+/PKC signaling pathway added of PTH1-34.
Results:(1) The Immunohistochemical staining showed PTHR1, CTR weak-medium expressed in normal thyroid, nodular goiter and thyroid papillary carcinoma and medullary thyroid carcinoma tissue. There was no significant difference between different tissue type group of thyroid tumors on PTHR1medium expression (p>0.05) and there was significant difference between different tissue type group of thyroid tumors on PTHR1strong positive expression (p<0.05).Expression of GRK2, GRK5was no difference between the normal thyroid and nodular goiter groups (p>0.05). GRK2expression was strong positive in papillary thyroid carcinoma and was weakly positive in medullary thyroid carcinoma tissue (p<0.05), GRK5expression were both weakly positive expression in papillary thyroid carcinoma and medullary thyroid carcinoma tissue. There was no significant difference between the two groups (p>0.05).Real-time fluorescence quantitative polymerase chain reaction showed there was no significant difference in the expression of the PTHR1mRNA CTR mRNA between the normal thyroid, nodular goiter and papillary thyroid carcinoma (P>0.05) but there was significant difference in the expression of the PTHR1mRNA CTR mRNA between the normal thyroid and medullary thyroid carcinoma (p<0.05). The expression of GRK2mRNA and GRK5mRNA was no significant difference between normal thyroid group and nodular goiter group (p>0.05). The expression of GRK2mRNA and GRK5mRNA in papillary thyroid carcinoma was significantly higher than normal thyroid group (p<0.05). GRK2mRNA expression was significantly higher than normal thyroid group and GRK5. mRNA expression was significantly lower than normal thyroid group in medullary thyroid carcinoma group which both showed the significant difference (p<0.05).Between normal thyroid, nodular goiter, thyroid papillary carcinoma of PKA mRNA and PKC mRNA expression was no significant difference (p>0.05); compared with normal thyroid group, the expression of PKA mRNA of PKC mRNA were significantly higher (p<0.05).There was no significant difference of expression CaM mRNA between normal thyroid, nodular goiter, thyroid papillary carcinoma (p>0.05).The expression CaM mRNA was higher in medullary thyroid carcinoma than normal thyroid that there was significant difference between two groups (p<0.05). Western blot showed same results as real-time fluorescence quantitative polymerase chain reaction.(2) Medullary thyroid carcinoma TT cells culture in F-12K medium showed long spindle, polygon or irregular shape to stick wall growth. It showed adherent growth of about80%of the48-to72hours after TT cells adherent fusion, give PTH1-34role significantly reduce cell number and some cells the cell body shrinkage and round cell gap is further widened, some cells showed growth delay, suspension cells and debris increased; given PTHR McAb to the role of cell number did not significantly reduce the Some cells showed a growth delay, cell morphology and vitality was no significant change. Add PTH1-34TT cell supernatant supernatant PTH levels significantly higher (p <0.05) and CT levels were significantly lower (p<0.05) between the different doses of PTH1-34TT supernatant level no significant difference (p>0.05).
Conclusions:(1) Thyroid C cell surface express PTHR1and CTR. Due to the high expression of the second messenger-regulated kinase (PKA and PKC) and CaM in medullary thyroid carcinoma tissue, G protein-coupled receptor kinase-GRK2and GRK5expression differently that GRK2activity enhance and GRK5activity weakened. As different phosphorylation of GRK2and GRK5on the thyroid C cell surface GPCRs, the result showed increase expression of PTHR1and CTR in medullary thyroid carcinoma tissue.(2) Add through the exogenous PTH1-34and the PTHR McAb, observe PTH1-34in vitro effects of medullary thyroid carcinoma TT cell line, cell culture supernatant showed that PTH levels were significantly increased CT levels; of cAMP concentration increased at the same time PKA rise in the PKC lower. At the same time CaM decreased expression of GRK2reduced expression of GRK5expression was elevated, and ultimately activate the AC-cAMP-PKA signaling system and inhibition of Ca2+/PKC signaling system signaling pathway conduction, resulting in medullary thyroid carcinoma TT cell growth inhibition, the study further revealed the PTHR1signaling pathway conduction growth of malignant cells, this study suggests that the PTHR1could become one of the targets of targeted therapy of medullary thyroid carcinoma, and opens up new perspectives and ideas for the treatment of medullary thyroid carcinoma.
引文
[1]Gilliland FD, Hunt WC, Morris DM, Key CR. Prognostic factors for thyroid carcinoma:a population-based study of 15,698 cases from the Surveillance, Epidemiology and End Results (SEER) program,1973-1991. [J]. Cancer. 1997,79(3):564-573.
[2]Hundahl SA, Fleming ID, Fremgen AM, Menck HR. A national cancer database report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985-1995. [J]. Cancer.1998,83(12):2638-2648..
[3]. Marsh DJ, Learoyd DL, Robinson BG. Medullary thyroid carcinoma:recent advances and management update. [J]. Thyroid,1995,5(5):407-424.
[4]. Dequanter D, Lothaire P. Medullary thyroid cancer:surgical results and prognostic factors. [J].Rev Med Liege.2010; 65(7-8):450-452.
[5], Dackiw AP. The surgical management of medullary thyroid cancer. [J]. Otolaryngol Clin North Am.2010; 43(2):365-374.
[6]Modigliani E, Cohen R, Campos JM, et al. Prognostic factors for survival and for biochemical cure in medullary thyroid carcinoma:results in 899 patients. The GETC Study Group. Groupe d'e'tude des tumeurs a'calcitonine. [J]. Clin Endocrinol 1998,48(3):265-73.
[7]Richard T. Kloos, Charis Eng, Douglas B. et al. Medullary thyroid cancer: management guidelines of the American thyroid association, thyroid. [J]. Thyroid,2009,19(6):565-612.
[8]. Traugott A, Moley JF. Medullary thyroid cancer:medical management and follow-up. [J].Curr Treat Options Oncol.2005,6(4):339-346.
[9]Wells SA, Gosnell JE, Gagel RF, et al. Vandetanib for the treatment of patients with locally advanced or metastatic hereditary medullary thyroid cancer.[J]. J Clin Oncol,2010,28(5):767-772.
[10]Asai N, Iwashita T, Matsuyama M, et al. Mechanism of activation of the RET proto2oncogene by Multiple Endocrine Neoplasia 2A mutations. [J]. Mol Cell Biol.1995,15(3):1613-1618
[11]Lin HY, Harris TL, Flannery MS, Aruffo A, Kaji EH, Gorn A, Kolakowski LF, Lodish HF, Goldring SR. Expression cloning of an adenylate cyclase-coupled calcitonin receptor. [J]. Science,1991,254(5034):1022-1024.
[12]Force T, Bonventre JV, Flannery MR, Gorn AH, Yamin M, Goldring SR. A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C. [J]. Am J Physiol,1992,262(6 Pt 2):F1110-1115
[13]Findlay DM, Michelangeli VP, Robinson PJ. Protein kinase- C -induced down regulation of calcitonin receptors and calcitonin activated adenylate cyclase in T47D and BEN cells. [J]. Endocrinology,1989,125(5):2656-2663.
[14]Suzuki K, Uchii M, Nozawa R. Expression of calcitonin receptors on human myeloid leukemia cells. [J]. J Biochem,1995,118(2):448-52.
[15]Rosenbaum DM, Rasmussen SG, Kobilka KB. The structure and function of G- protein- coupled receptors. [J]. Nature,2009,459(7245):356-363.
[16]Metaye T, Menet E, Guilhot J & Kraimps JL. Expression and activity of G protein-coupled receptor kinases in differentiated thyroid carcinoma.[J] Journal of Clinical Endocrinology and Metabolism.2002,87(7):3279-3286.
[17]Chase L R, A urbach G D. Parathyroid function and the renal secretion of 3', 5'-adenylic acid. [J] P roc Natl Acad Sci USA,1967,58(2):518-25.
[18]Moore CAC, Milano SK & Benovic JL 2007 Regulation of receptor trafficking by GRKs and arrestins. [J] Annu Rev Physiol.2007,69:451-482.
[19]Rapacciuolo A, Suvarna S, Barki2Harrington L, et al. Protein kinase A and G-protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways [J]. J Biol Chem, 2003,278 (37):35403-35411.
[20]谢毅,龚兴国,钟文涛.G蛋白偶联受体激酶的调控.[J].细胞生物学杂志2004,26：509-512
[21]Nagayama Y, Tanaka K, Namba H, Yamashita S & Niwa M. Expression and regulation of G protein-coupled receptor kinase 5 and β-arrestin-1 in rat thyroid FRTL5 cells. [J]. Thyroid,6(6):627-31.
[22]Metaye T, Menet E, Guilhot J & Kraimps JL 2002 Expression and activity of G protein-coupled receptor kinases in differentiated thyroid carcinoma. [J]. J Clin Endocrinol Metab.2002,87(7):3279-86.
[23]Voigt C, Holzapfel HP, Meyer S & Paschke R. Increased expression of G-protein-coupled receptor kinases 3 and 4 in hyperfunctioning thyroid nodules. [J]. J Endocrinol.2004,182(1):173-82.
[24]Swulius MT, Waxhan MN. Ca2+/calmodulin-dependent protein kinase. [J]. Cell Mol Life Sci.2008,65 (17):2637-2657.
[25]Couchonnal LF, Anderson ME. The role of calmodulin kinase Ⅱ in myocardial physiology and disease. [J].Physiology (Bethesda),2008,23: 151-159.
[26]Dicker F, Quitterer U, Winstel R, et al. Phosphorylation-independent inhibition of parathyroid hormone receptor signaling by G protein-coupled receptor kinases [J]. Proc Natl Acad Sci USA,1999,96 (10):5476-5481.
[27]Elorza A, Sarnago S, Mayor F. Agonist-dependent modulation of G protein-coupled receptor kinase 2 by mitogen-activated protein kinases [J].Mol Pharmacol,2000,57 (4):778-783.
[28]Audrey C, Stephane A L, Marc G C, et al. Endocytosis of G protein-coupled receptors:roles of G protein-coupled receptor kinases and beta-arrestin proteins [J].Prog Neurobiol,2002,66(2):61-79.
[29]Dequanter D, Lothaire P. Medullary thyroid cancer:surgical results and prognostic factors. [J].Rev Med Liege.2010; 65(7-8):450-452.
[30]Traugott A, Moley JF. Medullary thyroid cancer:medical management and follow-up. [J].Curr Treat Options Oncol.2005; 6(4):339-346.
[31]Moo-Young TA, Traugott AL, Moley JF. Sporadic and familial medullary thyroid carcinoma:state of the art.[J].Surg Clin North Am.2009; 89(5):1193-1204.
[32]Weber T, Shilling T, Buchler MW. Thyroid carcinoma. [J]. Curr Opin Oncol. 2006,18(1):30-35.
[33]Yasuhiro Ito, Akira Miyauchi, Tomonori Yabuta, et al. Alternative Surgical Strategies and Favorable Outcomes in Patients with Medullary Thyroid Carcinoma in Japan:Experience of a Single Institution[J].World J Surg.2009; 33(1):58-66.
[34]Chauvin S, Bencsik M, Bambino T and Nissenson RA. Parathyroid hormone receptor recycling:role of receptor dephosphorylation and p-arrestin. [J].Molecular Endocrinology.2002,16(12):2720-32.
[35]Turner PR, Mefford S, Christakos S, Nissenson RA. Apoptosis mediated by activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP). [J]. Mol Endocrinol.2000,14(2):241-54.
[36]Christian A. Molecular pathogenesis of MEN 2-associated tumors. [J]. Fam Cancer,2005,4(1):3-7.
[37]Kodama Y,Asai N, Kawai K, et al. The RET proto-oncogene:a molecular therapeutic target in thyroid cancer [J]. Cancer Sci,2005,96 (3):143-148.
[38]Groot JW, Links T, Plukker J, et al. RET as a diagnostic target in sporadic and hereditary endocrine tumors. [J]. Endocr Rev,2006,27 (5):535-560.
[39]Pigny P, Bauters C, Wermau JL, et al. A novel 9-base pair dup lication in RET exon 8 in familial medullary thyroid carcinoma. [J].J Clin Endocrinol Metab,1999,84 (5):1700-1704.
[40]Musholt TJ, Hanack J, Brehm C, et al. Searching for non RETmolecular alterations in medullary thyroid carcinoma exp ression analysis by mRNA differential disp lay [J]. World J Surg,2005,29(4):472-82.
[41]Ming G, Yang Y. Study of calcitonin and calcitonin gene-related peptide in patients with medullary thyroid carcinoma. [J]. Chin J Clin Oncol,2005,2(1): 500-504.
[42]Chauvin S, Bencsik M, Bambino T, Nissenson RA.. Parathyroid hormone receptor recycling:role of receptor dephosphorylation and β-arrestin. [J]. Mol Endocrinol.2002,16(12):2720-32.
[43]Romero G, Sneddon WB, Yang Y, et al. Parathyroid hormone receptor directly interacts with dishevelled to regulate beta-catenin signaling and osteoclastogenesis. [J]. J Biol Chem.2010,19(285):14756-14763.
[44]Chen X, Dai JC, Greenfield EM. Termination of immediate - early gene expression after stimulation by parathyroid hormone or isoproterenol. [J]. Am J Physiol Cell Physiol.2002,283(5):C1432-40.
[45]Metaye T, Levillain P, Kraimps JL and Perdrisot R.. Immunohistochemical detection, regulation and antiproliferative function of G-protein-coupled receptor kinase 2 in thyroid carcinomas. [J]. J Endocrinol.2008,198(1):101-10
[46]Vilardaga JP, Frank M, Krasel C, et al. Differential Conformational Requirements for Activation of G Proteins and the Regulatory Proteins Arrestin and G Protein-coupled Receptor Kinase in the G Protein-coupled Receptor for Parathyroid Hormone (PTH)/PTH-related Protein. [J]. J Biol Chem.2001, (276),36:33435-33443.
[2]Hundahl SA, Fleming ID, Fremgen AM, Menck HR. A national cancer database report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985-1995. [J]. Cancer.1998,83(12):2638-2648..
[3]. Marsh DJ, Learoyd DL, Robinson BG. Medullary thyroid carcinoma:recent advances and management update. [J]. Thyroid,1995,5(5):407-424.
[4]. Dequanter D, Lothaire P. Medullary thyroid cancer:surgical results and prognostic factors. [J].Rev Med Liege.2010; 65(7-8):450-452.
[5], Dackiw AP. The surgical management of medullary thyroid cancer. [J]. Otolaryngol Clin North Am.2010; 43(2):365-374.
[6]Modigliani E, Cohen R, Campos JM, et al. Prognostic factors for survival and for biochemical cure in medullary thyroid carcinoma:results in 899 patients. The GETC Study Group. Groupe d'e'tude des tumeurs a'calcitonine. [J]. Clin Endocrinol 1998,48(3):265-73.
[7]Richard T. Kloos, Charis Eng, Douglas B. et al. Medullary thyroid cancer: management guidelines of the American thyroid association, thyroid. [J]. Thyroid,2009,19(6):565-612.
[8]. Traugott A, Moley JF. Medullary thyroid cancer:medical management and follow-up. [J].Curr Treat Options Oncol.2005,6(4):339-346.
[9]Wells SA, Gosnell JE, Gagel RF, et al. Vandetanib for the treatment of patients with locally advanced or metastatic hereditary medullary thyroid cancer.[J]. J Clin Oncol,2010,28(5):767-772.
[10]Asai N, Iwashita T, Matsuyama M, et al. Mechanism of activation of the RET proto2oncogene by Multiple Endocrine Neoplasia 2A mutations. [J]. Mol Cell Biol.1995,15(3):1613-1618
[11]Lin HY, Harris TL, Flannery MS, Aruffo A, Kaji EH, Gorn A, Kolakowski LF, Lodish HF, Goldring SR. Expression cloning of an adenylate cyclase-coupled calcitonin receptor. [J]. Science,1991,254(5034):1022-1024.
[12]Force T, Bonventre JV, Flannery MR, Gorn AH, Yamin M, Goldring SR. A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C. [J]. Am J Physiol,1992,262(6 Pt 2):F1110-1115
[13]Findlay DM, Michelangeli VP, Robinson PJ. Protein kinase- C -induced down regulation of calcitonin receptors and calcitonin activated adenylate cyclase in T47D and BEN cells. [J]. Endocrinology,1989,125(5):2656-2663.
[14]Suzuki K, Uchii M, Nozawa R. Expression of calcitonin receptors on human myeloid leukemia cells. [J]. J Biochem,1995,118(2):448-52.
[15]Rosenbaum DM, Rasmussen SG, Kobilka KB. The structure and function of G- protein- coupled receptors. [J]. Nature,2009,459(7245):356-363.
[16]Metaye T, Menet E, Guilhot J & Kraimps JL. Expression and activity of G protein-coupled receptor kinases in differentiated thyroid carcinoma.[J] Journal of Clinical Endocrinology and Metabolism.2002,87(7):3279-3286.
[17]Chase L R, A urbach G D. Parathyroid function and the renal secretion of 3', 5'-adenylic acid. [J] P roc Natl Acad Sci USA,1967,58(2):518-25.
[18]Moore CAC, Milano SK & Benovic JL 2007 Regulation of receptor trafficking by GRKs and arrestins. [J] Annu Rev Physiol.2007,69:451-482.
[19]Rapacciuolo A, Suvarna S, Barki2Harrington L, et al. Protein kinase A and G-protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways [J]. J Biol Chem, 2003,278 (37):35403-35411.
[20]谢毅,龚兴国,钟文涛.G蛋白偶联受体激酶的调控.[J].细胞生物学杂志2004,26：509-512
[21]Nagayama Y, Tanaka K, Namba H, Yamashita S & Niwa M. Expression and regulation of G protein-coupled receptor kinase 5 and β-arrestin-1 in rat thyroid FRTL5 cells. [J]. Thyroid,6(6):627-31.
[22]Metaye T, Menet E, Guilhot J & Kraimps JL 2002 Expression and activity of G protein-coupled receptor kinases in differentiated thyroid carcinoma. [J]. J Clin Endocrinol Metab.2002,87(7):3279-86.
[23]Voigt C, Holzapfel HP, Meyer S & Paschke R. Increased expression of G-protein-coupled receptor kinases 3 and 4 in hyperfunctioning thyroid nodules. [J]. J Endocrinol.2004,182(1):173-82.
[24]Swulius MT, Waxhan MN. Ca2+/calmodulin-dependent protein kinase. [J]. Cell Mol Life Sci.2008,65 (17):2637-2657.
[25]Couchonnal LF, Anderson ME. The role of calmodulin kinase Ⅱ in myocardial physiology and disease. [J].Physiology (Bethesda),2008,23: 151-159.
[26]Dicker F, Quitterer U, Winstel R, et al. Phosphorylation-independent inhibition of parathyroid hormone receptor signaling by G protein-coupled receptor kinases [J]. Proc Natl Acad Sci USA,1999,96 (10):5476-5481.
[27]Elorza A, Sarnago S, Mayor F. Agonist-dependent modulation of G protein-coupled receptor kinase 2 by mitogen-activated protein kinases [J].Mol Pharmacol,2000,57 (4):778-783.
[28]Audrey C, Stephane A L, Marc G C, et al. Endocytosis of G protein-coupled receptors:roles of G protein-coupled receptor kinases and beta-arrestin proteins [J].Prog Neurobiol,2002,66(2):61-79.
[29]Dequanter D, Lothaire P. Medullary thyroid cancer:surgical results and prognostic factors. [J].Rev Med Liege.2010; 65(7-8):450-452.
[30]Traugott A, Moley JF. Medullary thyroid cancer:medical management and follow-up. [J].Curr Treat Options Oncol.2005; 6(4):339-346.
[31]Moo-Young TA, Traugott AL, Moley JF. Sporadic and familial medullary thyroid carcinoma:state of the art.[J].Surg Clin North Am.2009; 89(5):1193-1204.
[32]Weber T, Shilling T, Buchler MW. Thyroid carcinoma. [J]. Curr Opin Oncol. 2006,18(1):30-35.
[33]Yasuhiro Ito, Akira Miyauchi, Tomonori Yabuta, et al. Alternative Surgical Strategies and Favorable Outcomes in Patients with Medullary Thyroid Carcinoma in Japan:Experience of a Single Institution[J].World J Surg.2009; 33(1):58-66.
[34]Chauvin S, Bencsik M, Bambino T and Nissenson RA. Parathyroid hormone receptor recycling:role of receptor dephosphorylation and p-arrestin. [J].Molecular Endocrinology.2002,16(12):2720-32.
[35]Turner PR, Mefford S, Christakos S, Nissenson RA. Apoptosis mediated by activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP). [J]. Mol Endocrinol.2000,14(2):241-54.
[36]Christian A. Molecular pathogenesis of MEN 2-associated tumors. [J]. Fam Cancer,2005,4(1):3-7.
[37]Kodama Y,Asai N, Kawai K, et al. The RET proto-oncogene:a molecular therapeutic target in thyroid cancer [J]. Cancer Sci,2005,96 (3):143-148.
[38]Groot JW, Links T, Plukker J, et al. RET as a diagnostic target in sporadic and hereditary endocrine tumors. [J]. Endocr Rev,2006,27 (5):535-560.
[39]Pigny P, Bauters C, Wermau JL, et al. A novel 9-base pair dup lication in RET exon 8 in familial medullary thyroid carcinoma. [J].J Clin Endocrinol Metab,1999,84 (5):1700-1704.
[40]Musholt TJ, Hanack J, Brehm C, et al. Searching for non RETmolecular alterations in medullary thyroid carcinoma exp ression analysis by mRNA differential disp lay [J]. World J Surg,2005,29(4):472-82.
[41]Ming G, Yang Y. Study of calcitonin and calcitonin gene-related peptide in patients with medullary thyroid carcinoma. [J]. Chin J Clin Oncol,2005,2(1): 500-504.
[42]Chauvin S, Bencsik M, Bambino T, Nissenson RA.. Parathyroid hormone receptor recycling:role of receptor dephosphorylation and β-arrestin. [J]. Mol Endocrinol.2002,16(12):2720-32.
[43]Romero G, Sneddon WB, Yang Y, et al. Parathyroid hormone receptor directly interacts with dishevelled to regulate beta-catenin signaling and osteoclastogenesis. [J]. J Biol Chem.2010,19(285):14756-14763.
[44]Chen X, Dai JC, Greenfield EM. Termination of immediate - early gene expression after stimulation by parathyroid hormone or isoproterenol. [J]. Am J Physiol Cell Physiol.2002,283(5):C1432-40.
[45]Metaye T, Levillain P, Kraimps JL and Perdrisot R.. Immunohistochemical detection, regulation and antiproliferative function of G-protein-coupled receptor kinase 2 in thyroid carcinomas. [J]. J Endocrinol.2008,198(1):101-10
[46]Vilardaga JP, Frank M, Krasel C, et al. Differential Conformational Requirements for Activation of G Proteins and the Regulatory Proteins Arrestin and G Protein-coupled Receptor Kinase in the G Protein-coupled Receptor for Parathyroid Hormone (PTH)/PTH-related Protein. [J]. J Biol Chem.2001, (276),36:33435-33443.