Transmembrane helix M6 in sarco(endo)plasmic reticulum Ca2+-ATPase forms a functional interaction site with phospholamban -: Evidence for physical, interactions at other sites
Transmembrane helix M6 in sarco(endo)plasmic reticulum Ca2+-ATPase forms a functional interaction site with phospholamban -: Evidence for physical, interactions at other sites
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DOI：
10.1074/jbc.274.46.32855
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发表时间：
1999-11-12
影响因子：
4.8
通讯作者：
MacLennan, DH
中科院分区：
文献类型：
--
作者：
Asahi, M;Kimura, Y;MacLennan, DH
在早期的研究中（Kimura，Y.，Kurzydlowski，K.，多田，M.，和MacLennan，D. H.（1997）J.Biol.Chem.272，15061 - 15064），受磷蛋白（PLN）跨膜螺旋的一面上的氨基酸突变导致PLN对肌（内）质网Ca 2 +-ATP酶（SERCA）分子的抑制作用丧失。该螺旋面被提出形成PLN与SERCA分子中的跨膜螺旋相互作用的位点。为了确定SERCA1a中预测的跨膜螺旋M4、M5、M6或Mg是否与PLN相互作用，SERCA1a突变体与野生型PLN共表达，并测量对Ca2+转运的Ca2+依赖性的影响。野生型抑制性相互作用使SERCA1a的表观Ca2+亲和力平均偏移-0.34 pCa单位，但M4中7个突变中的4个导致表观Ca2+亲和力的抑制性偏移更大，平均偏移-0.53 pCa单位。M5中的7个突变导致-0.32 pCa单位的平均偏移，M8中的7个突变导致-0.30 pCa单位的平均偏移。在M6的11个突变中，1，Q791A，增加了抑制位移（-0.59 pCa单位）和5，V795A（-0.11），L802A（-0.07），L802V（-0.04），T805A（-0.11）和F809A（-0.12），降低了抑制性偏移，与位于预测的M6螺旋的一面上的瓦尔（795）、Leu（802）、Thr（805）和Phe（809）的观点一致，在SERCA1a中形成与PLN相互作用的位点。增强PLN抑制功能的SERCA1a的M4、M6或M8中的那些突变没有增强PLN与SERCA1a的物理结合，但是M6中的突变体V795A和L802A降低了PLN抑制功能，降低了物理结合，如通过免疫共沉淀所测量的。在相关的研究中，那些获得抑制功能的PLN突变体也增加了野生型SERCAla的免疫共沉淀水平，而那些失去抑制功能的突变体也减少了关联，将功能相互作用位点与物理相互作用位点相关联。因此，功能和物理数据都证实PLN与M6 SERCAla相互作用。
In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), mutation of amino acids on one face of the phospholamban (PLN) transmembrane helix led to loss of PLN inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) molecules. This helical face was proposed to form a site of PLN interaction with a transmembrane helix in SERCA molecules. To determine whether predicted transmembrane helices M4, M5, M6, or Mg in SERCA1a interact with PLN, SERCA1a mutants were co expressed with wild-type PLN and effects on Ca2+ dependence of Ca2+ transport were measured. Wild-type inhibitory interactions shifted apparent Ca2+ affinity of SERCA1a by an average of -0.34 pCa units, but four of the seven mutations in M4 led to a more inhibitory shift in apparent Ca2+ affinity, averaging -0.53 pCa units. Seven mutations in M5 led to an average shift of -0.32 pCa units and seven mutations in M8 led to an average shift of -0.30 pCa units. Among 11 mutations in M6, 1, Q791A, increased the inhibitory shift (-0.59 pCa units) and 5, V795A (-0.11), L802A (-0.07), L802V (-0.04), T805A (-0.11), and F809A (-0.12), reduced the inhibitory shift, consistent with the view that Val(795), Leu(802), Thr(805), and Phe(809), located on one face of a predicted M6 helix, form a site in SERCA1a for interaction with PLN. Those mutations in M4, M6, or M8 of SERCA1a that enhanced PLN inhibitory function did not enhance PLN physical association with SERCA1a, but mutants V795A and L802A in M6, which decreased PLN inhibitory function, decreased physical association, as measured by co-immunoprecipitation. In related studies, those PLN mutants that gained inhibitory function also increased levels of co-immunoprecipitation of wild-type SERCAla and those that lost inhibitory function also reduced association, correlating functional interaction sites with physical interaction sites. Thus, both functional and physical data confirm that PLN interacts with M6 SERCAla.