Publication: Eritroid hücrelerde G protein ekspresyonunun incelenmesi
Abstract
?, ß ve ? alt birimlerinden oluşan heterotrimerik G proteinleri, hücre dışı sinyallerin fosfolipaz C, adenilat siklaz ve iyon kanalları gibi efektörlere ulaştırılmasına aracılık eder. Bu çalışma, G proteinlerinin hücre farklılaşmasında da rol oynadığını düşündüren çalışmalar ışığında, eritroid hücre farklılaşmasında G proteinlerinin olası rolünü incelemeyi amaçlamaktadır. Hemin ile farklılaşmaya indüklenen K562 hücrelerinden ham zar kesimleri elde edildi ve bu kesimlerde GTP bağlanma tepkimesinin ligand ve protein miktarına bağımlılığı gösterildi. Kd ve Bmax değerleri saptandı ve GTP?S bağlanmasının GTP ile baskılandığı gösterildi. K562 hücre farklılaşması sürecinde G protein ekspresyonunda olası bir değişikliği saptamak üzere, hücre zar kesimlerinin Gortak?, Gß?, Go?, Gi12?, Gs?, Gq? ve G16?' ya özgün antikorlarla özgün tepkimesi incelendi. G protein ? alt birimlerinin tümünü tanıyan antikor ile elde edilen özgün sinyalin K (Kontrol) grubunda, F(Farklılaşmış) grubuna göre çok daha kuvvetli olduğu ancak, ß? alt birim miktarları açısından önemli bir fark olmadığı gözlendi. Beyin zar proteinlerinin % 1-2' ini oluşturan Go? proteinin K562 hücrelerinde bulunmadığı belirlendi. Farklılaşmış hücrelerdeki Gs?-L düzeyinin kontrollere kıyasla beşinci günde % 50, altıncı günde % 19 oranında arttığı, Gs?-S düzeyinin ise beşinci günde % 93, altıncı günde % 41 oranında arttığı ayrıca kısa formun daha fazla miktarda olduğu gözlendi. Gs? ekspresyonu RNA düzeyinde saptandı. Gs?-L' e ait sinyalin, Western-blot analizinde olduğu gibi Gs?-S' e göre daha zayıf olması, K562 hücrelerinde Gs? ' nın kısa formunun daha yüksek düzeyde ekspres edildiğini doğruladı. K562 hücrelerinde Gi1? ekspresyonu belirlenmedi, Gi2? düzeylerinin ise farklılaşma ile % 12 oranında azaldığı gözlendi. Ayrıca fosfolipaz C yolaklarının düzenlenmesine aracılık eden Gq/ 11? düzeylerinin farklılaşmaya koşut olarak % 22 oranında arttığı belirlendi. Hematopoietik hücrelerde ekspres edilen G16? proteinine karşı geliştirilen AS 339 antikoru ile etkileşim saptanamadı. Sonuç olarak, Gs?, Gi2? ve Gq?' nın K562 farklılaşma sürecinde değişen düzeyleri bir çok sistemde olduğu gibi eritroid hücre farklılaşmasında da G proteinlerinin katkısı olduğunu gösterdi.
Heterotrimeric G proteins which are composed of ?, ß and ? subunits couple extracellular signals to intracellular effectors such as phospholipase C, adenylate cyclase and ion channels. Based on previous studies that demonstrate the role of G proteins in cell differentiation, this study was designed to investigate the possible role of G proteins in erythroid cell differentiation. K562 cells grown in suspension culture were induced to differentiate with hemin and centrifuged to obtain crude cell membranes. Guanine nucleotide binding activity was assayed in crude membrane fractions. GTP?S binding was dependent on ligand and membrane protein concentration; Kd and Bmax values for the reaction were determined, and GTP?S binding was demonstrated to be inhibited by GTP. Various cell fractions obtained from differentiated and nondifferentiated cells were analyzed for G protein ? and ß? subunits by Western blot analysis, using antibodies specific for Gcommon?,, Gß?, Go?, Gi12?, Gs?, Gq? and G16?. Immuno-reactivity with anti-Gs? common antibody was stronger in differentiated cells than in their nondifferentiated counterparts, but the ß? signals were similar in both groups. Go? which constitutes about 1-2 % of brain membranes was absent in K562 cells. The level of the long form of Gs? in differentiated cells was found to be 50 % higher and 19 % higher than the level in the nondifferentiated group, on the fifth and sixth days after induction, respectively. Compared to the control group; the short form of Gs? was found to be 93 % higher in the fifth day and 41 % on the sixth day. Furthermore, the content of the long form of Gs? was lower than that of Gs?-S. This finding was confirmed by Northern blot analysis. Upon differentiation, the levels of Gi2? were found to decrease by 12 % whereas those of Gq/ 11?, the G protein coupled to the phosphplipase C pathway, increased by 22 %. Gi1? and G16? could not be determined by immunoblot analysis. In conclusion, Gs?, Gi2?, Gq? and Gß? expression was detected in K562 cells. The change in the levels of Gs?, Gi2? and Gq? during K562 differentiation implies that G proteins are also involved in erythroid cell differentiation.
Heterotrimeric G proteins which are composed of ?, ß and ? subunits couple extracellular signals to intracellular effectors such as phospholipase C, adenylate cyclase and ion channels. Based on previous studies that demonstrate the role of G proteins in cell differentiation, this study was designed to investigate the possible role of G proteins in erythroid cell differentiation. K562 cells grown in suspension culture were induced to differentiate with hemin and centrifuged to obtain crude cell membranes. Guanine nucleotide binding activity was assayed in crude membrane fractions. GTP?S binding was dependent on ligand and membrane protein concentration; Kd and Bmax values for the reaction were determined, and GTP?S binding was demonstrated to be inhibited by GTP. Various cell fractions obtained from differentiated and nondifferentiated cells were analyzed for G protein ? and ß? subunits by Western blot analysis, using antibodies specific for Gcommon?,, Gß?, Go?, Gi12?, Gs?, Gq? and G16?. Immuno-reactivity with anti-Gs? common antibody was stronger in differentiated cells than in their nondifferentiated counterparts, but the ß? signals were similar in both groups. Go? which constitutes about 1-2 % of brain membranes was absent in K562 cells. The level of the long form of Gs? in differentiated cells was found to be 50 % higher and 19 % higher than the level in the nondifferentiated group, on the fifth and sixth days after induction, respectively. Compared to the control group; the short form of Gs? was found to be 93 % higher in the fifth day and 41 % on the sixth day. Furthermore, the content of the long form of Gs? was lower than that of Gs?-S. This finding was confirmed by Northern blot analysis. Upon differentiation, the levels of Gi2? were found to decrease by 12 % whereas those of Gq/ 11?, the G protein coupled to the phosphplipase C pathway, increased by 22 %. Gi1? and G16? could not be determined by immunoblot analysis. In conclusion, Gs?, Gi2?, Gq? and Gß? expression was detected in K562 cells. The change in the levels of Gs?, Gi2? and Gq? during K562 differentiation implies that G proteins are also involved in erythroid cell differentiation.