Salim Samina, Sinnarajah Srikumar, Kehrl John H, Dessauer Carmen W
Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, 6431 Fannin Street, Houston, TX 77030, USA.
J Biol Chem. 2003 May 2;278(18):15842-9. doi: 10.1074/jbc.M210663200. Epub 2003 Feb 25.
The production of cAMP is controlled on many levels, notably at the level of cAMP synthesis by the enzyme adenylyl cyclase. We have recently identified a new regulator of adenylyl cyclase activity, RGS2, which decreases cAMP accumulation when overexpressed in HEK293 cells and inhibits the in vitro activity of types III, V, and VI adenylyl cyclase. In addition, RGS2 blocking antibodies lead to elevated cAMP levels in olfactory neurons. Here we examine the nature of the interaction between RGS2 and type V adenylyl cyclase. In HEK293 cells expressing type V adenylyl cyclase, RGS2 inhibited Galpha(s)-Q227L- or beta(2)-adrenergic receptor-stimulated cAMP accumulation. Deletion of the N-terminal 19 amino acids of RGS2 abolished its ability to inhibit cAMP accumulation and to bind adenylyl cyclase. Further mutational analysis indicated that neither the C terminus, RGS GAP activity, nor the RGS box domain is required for inhibition of adenylyl cyclase. Alanine scanning of the N-terminal amino acids of RGS2 identified three residues responsible for the inhibitory function of RGS2. Furthermore, we show that RGS2 interacts directly with the C(1) but not the C(2) domain of type V adenylyl cyclase and that the inhibition by RGS2 is independent of inhibition by Galpha(i). These results provide clear evidence for functional effects of RGS2 on adenylyl cyclase activity that adds a new dimension to an intricate signaling network.
环磷酸腺苷（cAMP）的产生在多个层面受到调控，尤其是在由腺苷酸环化酶合成cAMP的层面。我们最近鉴定出一种腺苷酸环化酶活性的新调节因子RGS2，当它在人胚肾293（HEK293）细胞中过表达时，会减少cAMP的积累，并抑制III型、V型和VI型腺苷酸环化酶的体外活性。此外，RGS2阻断抗体可导致嗅觉神经元中的cAMP水平升高。在此，我们研究RGS2与V型腺苷酸环化酶之间相互作用的本质。在表达V型腺苷酸环化酶的HEK293细胞中，RGS2抑制Gαs - Q227L或β2 - 肾上腺素能受体刺激的cAMP积累。缺失RGS2的N端19个氨基酸消除了其抑制cAMP积累和结合腺苷酸环化酶的能力。进一步的突变分析表明，腺苷酸环化酶的抑制作用既不需要C端、RGS GAP活性，也不需要RGS盒结构域。对RGS2 N端氨基酸进行丙氨酸扫描鉴定出三个负责RGS2抑制功能的残基。此外，我们表明RGS2直接与V型腺苷酸环化酶的C1结构域相互作用，而不与C2结构域相互作用，并且RGS2的抑制作用独立于Gαi的抑制作用。这些结果为RGS2对腺苷酸环化酶活性的功能影响提供了明确证据，这为复杂的信号网络增添了新的维度。
