Ethier J F, Lussier J G, Silversides D W
Centre de Recherche en Reproduction Animale (CRRA), Department of Veterinary Biomedicine, Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Québec, Canada.
Endocrinology. 1997 Jun;138(6):2425-34. doi: 10.1210/endo.138.6.5173.
Activins are implicated in a variety of biological effects, particularly in reproductive processes such as embryonic development and folliculogenesis. Breakthroughs in the elucidation of the activin signal transduction mechanism were achieved with the characterization of the activin receptors, and the recent identification of cytoplasmic factors apparently involved in the signaling process. The present studies were undertaken to further analyze the activin signaling pathway. The complementary DNA coding for the bovine activin receptor type IIB (bActRIIB) was amplified by RT-PCR from corpus luteum and pituitary RNA, and cloned to characterize its role in activin signal transduction. Two complementary DNA isoforms (bActRIIB2 and bActRIIB5) were detected, coding for 512 amino acids and 498 amino acids, respectively. The shortest isoform lacked a sequence encoding a 14-amino acid stretch very rich in proline residues, located between the transmembrane region and the intracellular kinase domain. Intron sequencing and ribonuclease protection assay demonstrated that alternative splicing is responsible for the generation of these bActRIIB isoforms. This alternative splicing event is unique in that it has not been observed in other species, including the mouse, in which extensive alternative splicing of the ActRIIB messenger RNA is described. Comparison of this alternative sequence with other known proline-rich sequences showed that it has characteristics of a Src-homology 3 domain (SH3) binding site. Coprecipitation experiments have identified two proteins of 69 kDa and 71 kDa from an uterine endometrial cell line, specifically interacting with the short bActRIIB alternative proline-rich sequence. These results suggest that bActRIIB could have a protein-protein interaction, through its putative SH3 binding site, with at least two intracellular SH3-containing proteins.
激活素参与多种生物学效应，尤其在生殖过程中，如胚胎发育和卵泡发生。随着激活素受体的鉴定以及最近对明显参与信号传导过程的细胞质因子的识别，激活素信号转导机制的阐明取得了突破。本研究旨在进一步分析激活素信号通路。通过逆转录聚合酶链反应（RT-PCR）从黄体和垂体RNA中扩增出编码牛IIB型激活素受体（bActRIIB）的互补DNA，并进行克隆以表征其在激活素信号转导中的作用。检测到两种互补DNA异构体（bActRIIB2和bActRIIB5），分别编码512个氨基酸和498个氨基酸。最短的异构体缺少一段编码富含脯氨酸残基的14个氨基酸序列，该序列位于跨膜区和细胞内激酶结构域之间。内含子测序和核糖核酸酶保护试验表明，可变剪接是这些bActRIIB异构体产生的原因。这种可变剪接事件是独特的，因为在包括小鼠在内的其他物种中未观察到，在小鼠中描述了ActRIIB信使RNA的广泛可变剪接。将该可变序列与其他已知的富含脯氨酸序列进行比较，发现它具有Src同源3结构域（SH3）结合位点的特征。共沉淀实验从子宫子宫内膜细胞系中鉴定出两种分子量分别为69 kDa和71 kDa的蛋白质，它们与短的bActRIIB可变富含脯氨酸序列特异性相互作用。这些结果表明，bActRIIB可能通过其假定的SH3结合位点与至少两种细胞内含有SH3的蛋白质发生蛋白质-蛋白质相互作用。
