Markovic Danijela, Grammatopoulos Dimitris K
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.
Methods Mol Biol. 2010;634:285-307. doi: 10.1007/978-1-60761-652-8_21.
Mammalian adaptation to stressful stimuli requires activation of the type 1 corticotropin releasing hormone (CRH) receptor (CRH-R1), a 415 amino acid protein that belongs to the large superfamily of 7 transmembrane domain receptors that relay signals across cells through activation of G-proteins. CRH-R1 expression and activity is regulated at the gene level by mRNA alternative splicing that results in a number of CRH-R1 variants. This process can generate putative CRH-R1 receptor variants with distinct structural and signaling properties; their study can provide important insights about the structural determinants of CRH-R1 functional characteristics. Using site-directed mutagenesis by overlap extension polymerase chain reaction (OE-PCR), we investigated the structure-function relationships of a CRH-R1 variant with an extended 1st intracellular loop (IC1) (CRH-R1beta), a sequence modification that impairs signaling activity (such as cAMP production and MAPK activation). We identified a penta-amino acid cassette within the 29-amino acid insert of CRH-R1beta rich in positive charged amino acids (F(170)-R(174)), as an important structural determinant for the impaired cAMP response.
哺乳动物对应激刺激的适应需要激活1型促肾上腺皮质激素释放激素（CRH）受体（CRH-R1），这是一种由415个氨基酸组成的蛋白质，属于7跨膜结构域受体的大型超家族，该家族通过激活G蛋白在细胞间传递信号。CRH-R1的表达和活性在基因水平上受mRNA可变剪接的调控，可变剪接会产生多种CRH-R1变体。这一过程可产生具有不同结构和信号特性的假定CRH-R1受体变体；对它们的研究可为CRH-R1功能特性的结构决定因素提供重要见解。我们通过重叠延伸聚合酶链反应（OE-PCR）进行定点诱变，研究了一种具有延长的第一细胞内环（IC1）的CRH-R1变体（CRH-R1β）的结构-功能关系，这种序列修饰会损害信号活性（如cAMP产生和MAPK激活）。我们在富含带正电荷氨基酸的CRH-R1β的29个氨基酸插入片段中鉴定出一个五氨基酸盒（F(170)-R(174)），作为cAMP反应受损的重要结构决定因素。
