Potter L R, Garbers D L
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232.
J Biol Chem. 1994 May 20;269(20):14636-42.
Stimulation of guanylyl cyclase A (GC-A) by atrial natriuretic peptide (ANP) is antagonized by activators of protein kinase C (PKC). Thus, it has been suggested that PKC phosphorylates and desensitizes GC-A. Here, we have developed stable GC-A transfectants of NIH3T3 cells, which display marked reductions in hormone-dependent cGMP elevations and guanylyl cyclase activity after incubation with ANP or phorbol 12-myristate 13-acetate (PMA). ANP binding and immunoblot analysis indicated that the decreases were not due to receptor internalization or degradation. GC-A isolated from 32PO4-labeled cells contained phosphoserine and phosphothreonine. ANP and/or PMA addition caused substantial decreases in the 32P content of the receptor that coincided with reductions in hormone-dependent guanylyl cyclase activity. The specific PKC inhibitor, GF-109203X, completely blocked the PMA-dependent dephosphorylation and desensitization of GC-A but failed to inhibit either ANP-dependent process. Tryptic phosphopeptide maps of GC-A isolated from ANP- or PMA-treated cells were unique, suggesting that the sites that dephosphorylated in response to each agent were different. In contrast to previous reports, we conclude that PMA and ANP desensitization of GC-A are distinct events mediated by dephosphorylation of specific residues through PKC-dependent and -independent pathways, respectively.
心房利钠肽（ANP）对鸟苷酸环化酶A（GC-A）的刺激作用被蛋白激酶C（PKC）激活剂所拮抗。因此，有人提出PKC使GC-A磷酸化并使其脱敏。在此，我们构建了NIH3T3细胞的稳定GC-A转染子，在与ANP或佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）孵育后，其激素依赖性cGMP升高和鸟苷酸环化酶活性显著降低。ANP结合和免疫印迹分析表明，这种降低并非由于受体内化或降解所致。从32P标记的细胞中分离出的GC-A含有磷酸丝氨酸和磷酸苏氨酸。添加ANP和/或PMA导致受体的32P含量大幅下降，这与激素依赖性鸟苷酸环化酶活性的降低相一致。特异性PKC抑制剂GF-109203X完全阻断了PMA依赖性的GC-A去磷酸化和脱敏，但未能抑制任何一种ANP依赖性过程。从ANP或PMA处理的细胞中分离出的GC-A的胰蛋白酶磷酸肽图谱是独特的，这表明对每种试剂去磷酸化的位点是不同的。与先前的报道相反，我们得出结论，GC-A的PMA和ANP脱敏是不同的事件，分别通过PKC依赖性和非依赖性途径由特定残基的去磷酸化介导。
