Gros Robert, Ding Qingming, Chorazyczewski Jozef, Pickering J Geoffrey, Limbird Lee E, Feldman Ross D
Cell Biology Research Group, Robarts Research Institute, 100 Perth Dr, London, ON N6A 5K8, Canada.
Circ Res. 2006 Oct 13;99(8):845-52. doi: 10.1161/01.RES.0000245189.21703.c0. Epub 2006 Sep 14.
Compartmentation of cAMP signaling been demonstrated to be attributable to the structural association of protein kinase A (PKA) (via association with A-kinase anchoring proteins [AKAPs]) with phosphodiesterase and AKAP-dependent effector molecules. However, other mechanisms contributing to compartmentalization have not been rigorously explored, including the possibility that different isoforms of adenylyl cyclase (AC) may be functionally "compartmentalized" because of differential association with tethering or signaling molecules. To this end, we examined the effect of adenoviral transduction of representative AC isoforms (AC1, AC2, AC5, and AC6) on cellular cAMP production, PKA activation, extracellular signal-regulated kinase (ERK) activation, cell doubling and proliferation, as well as arborization responses (an index of cAMP-mediated cytoskeletal re-organization) in vascular smooth muscle cells. When isoforms were expressed at levels to achieve comparable forskolin-stimulated AC activity, only gene transfer of AC6 significantly enhanced PKA-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation and arborization responses. Treatment of control cells, which express AC6 endogenously, as well as vascular smooth overexpressing the AC6 isoform with small interfering RNA directed against AC6, significantly suppressed both isoproterenol-stimulated cAMP accumulation and arborization. Notably, the selective effects of AC6 expression were abrogated in the presence of phosphodiesterase suppression. In contrast, only the expression of AC1 enhanced forskolin-stimulated association of ERK with AC, demonstrated by coimmuno-isolation of ERK with Flag-tagged AC1, but not with Flag-tagged AC6. To determine whether these isoform-selective effects of AC were unique to differentiated and morphologically compartmentalized vascular smooth muscle cells or were a general property of these isoforms, we examined the consequence of expression of these various isoforms in human embryonic kidney (HEK) cells. Indeed, we observed similar isoform-dependent association of AC1 with ERK, activation of ERK by stimulation of AC1 with forskolin, and AC1-dependent lengthening of doubling time, indicating that these properties of AC1 are cell autologous and likely result from AC1-dependent protein-protein interactions. In aggregate, these findings suggest that isoform-selective signaling complexes likely contribute to various functional consequences of cAMP elevation in vascular smooth muscle cells.
环磷酸腺苷（cAMP）信号的区室化已被证明归因于蛋白激酶A（PKA）（通过与A激酶锚定蛋白[AKAPs]结合）与磷酸二酯酶和AKAP依赖性效应分子的结构关联。然而，尚未对导致区室化的其他机制进行严格探索，包括不同亚型的腺苷酸环化酶（AC）可能由于与拴系或信号分子的差异结合而在功能上“区室化”的可能性。为此，我们研究了代表性AC亚型（AC1、AC2、AC5和AC6）的腺病毒转导对血管平滑肌细胞中细胞cAMP产生、PKA激活、细胞外信号调节激酶（ERK）激活、细胞倍增和增殖以及树突化反应（cAMP介导的细胞骨架重组指标）的影响。当各亚型以实现可比的福斯可林刺激的AC活性水平表达时，只有AC6的基因转移显著增强了PKA依赖性血管舒张剂刺激的磷蛋白（VASP）磷酸化和树突化反应。用针对AC6的小干扰RNA处理内源性表达AC6的对照细胞以及过表达AC6亚型的血管平滑肌细胞，显著抑制了异丙肾上腺素刺激的cAMP积累和树突化。值得注意的是，在磷酸二酯酶抑制存在的情况下，AC6表达的选择性作用被消除。相反，只有AC1的表达增强了福斯可林刺激的ERK与AC的结合，通过用Flag标记的AC1而非Flag标记的AC6进行共免疫分离来证明。为了确定AC的这些亚型选择性作用是否是分化且形态区室化的血管平滑肌细胞所特有的，还是这些亚型的一般特性，我们研究了在人胚肾（HEK）细胞中表达这些不同亚型的后果。确实，我们观察到AC1与ERK有类似的亚型依赖性结合、用福斯可林刺激AC1激活ERK以及AC1依赖性的倍增时间延长，表明AC1的这些特性是细胞自身的，可能是由AC1依赖性的蛋白质 - 蛋白质相互作用导致的。总的来说，这些发现表明亚型选择性信号复合物可能促成了血管平滑肌细胞中cAMP升高的各种功能后果。
