Natochin M, McEntaffer R L, Artemyev N O
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
J Biol Chem. 1998 Mar 20;273(12):6731-5. doi: 10.1074/jbc.273.12.6731.
Members of the RGS family serve as GTPase-activating proteins (GAPs) for heterotrimeric G-proteins and negatively regulate signaling via G-protein-coupled receptors. The recently resolved crystal structure of RGS4 bound to Gialpha1 suggests two potential mechanisms for the GAP activity of RGS proteins as follows: stabilization of the Gialpha1 switch regions by RGS4 and the catalytic action of RGS4 residue Asn128. To elucidate a role of the Asn residue for RGS GAP function, we have investigated effects of the synthetic peptide corresponding to the Galpha binding domain of human retinal RGS (hRGSr) containing the key Asn at position 131, and we have carried out mutational analysis of Asn131. Synthetic peptide hRGSr-(123-140) retained its ability to bind the AlF4--complexed transducin alpha-subunit, Gtalpha.AlF4-, but failed to elicit stimulation of Gtalpha GTPase activity. Wild-type hRGSr stimulated Gtalpha GTPase activity by approximately 10-fold with an EC50 value of 100 nM. Mutant hRGSr proteins with substitutions of Asn131 by Ser and Gln had a significantly reduced affinity for Gtalpha but were capable of substantial stimulation of Gtalpha GTPase activity, 80 and 60% of Vmax, respectively. Mutants hRGSr-Leu131, hRGSr-Ala131, and hRGSr-Asp131 were able to accelerate Gtalpha GTPase activity only at very high concentrations (>10 microM) which appears to correlate with a further decrease of their affinity for transducin. Two mutants, hRGSr-His131 and hRGSr-Delta131, had no detectable binding to transducin. Mutational analysis of Asn131 suggests that the stabilization of the G-protein switch regions rather than catalytic action of the Asn residue is a key component for the RGS GAP action.
RGS家族成员作为异源三聚体G蛋白的GTP酶激活蛋白（GAP），通过G蛋白偶联受体对信号传导起负调控作用。最近解析的与Gialpha1结合的RGS4晶体结构提示了RGS蛋白GAP活性的两种潜在机制如下：RGS4对Gialpha1开关区域的稳定作用以及RGS4第128位天冬酰胺残基的催化作用。为阐明天冬酰胺残基在RGS GAP功能中的作用，我们研究了对应于人视网膜RGS（hRGSr）Galpha结合结构域且在第131位含有关键天冬酰胺的合成肽的作用，并对第131位天冬酰胺进行了突变分析。合成肽hRGSr-(123 - 140)保留了其与AlF4-复合转导蛋白α亚基Gtalpha.AlF4-结合的能力，但未能引发对Gtalpha GTP酶活性的刺激。野生型hRGSr刺激Gtalpha GTP酶活性约10倍，EC50值为100 nM。用丝氨酸和谷氨酰胺取代第131位天冬酰胺的突变型hRGSr蛋白对Gtalpha的亲和力显著降低，但能够分别以Vmax的80%和60%对Gtalpha GTP酶活性进行实质性刺激。突变体hRGSr-Leu131、hRGSr-Ala131和hRGSr-Asp131仅在非常高的浓度（>10 microM）下才能加速Gtalpha GTP酶活性，这似乎与其对转导蛋白亲和力的进一步降低相关。两个突变体hRGSr-His131和hRGSr-Delta131与转导蛋白没有可检测到的结合。对第131位天冬酰胺的突变分析表明，G蛋白开关区域的稳定作用而非天冬酰胺残基的催化作用是RGS GAP作用的关键组成部分。
