Patel Y C, Panetta R, Escher E, Greenwood M, Srikant C B
Fraser Laboratories, McGill University, Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada.
J Biol Chem. 1994 Jan 14;269(2):1506-9.
The pattern of expression of somatostatin receptor (SSTR) genes and gene products in AtT-20 cells was characterized in an attempt to explain the SST-28 binding selectivity that typifies these cells. AtT-20 cells expressed multiple SSTR mRNAs. Paradoxically, this included mRNA for three of the four SST-14 selective receptors: SSTR2 ( +), SSTR1 (+), SSTR4 (+). The SST-28 selective SSTR5 was expressed as a 3.8-kilobase (kb) transcript of relatively low abundance (+) in contrast to normal mouse pituitary which displayed high levels ( ) of a 2.4-kb SSTR5 mRNA. Immunoblot analysis of solubilized membranes with an antipeptide SSTR2 antibody revealed a single SSTR2 protein of 72 +/- 2 kDa. Preincubation of AtT-20 cell membranes with SSTR2 antibody reduced 125I-[Leu8,D-Trp22,Tyr25]SST-28 binding sites by 38%. Residual binding sites exhibited a 4.9-fold increase in affinity for SST-28, a 2.6-fold decrease in affinity for SST-14, and an SST-28:SST-14 potency ratio of 40:1 compared with a potency ratio of 3.5:1 in control membranes. These results demonstrate the expression of four SSTR genes in AtT-20 cells of which SSTR2 predominates. Blockade of SSTR2 with antibody exposes high affinity SST-28 selective sites with comparable binding characteristics to those reported for cloned SSTR5. These SST-28 binding sites may arise from a SSTR5 variant encoded by a high molecular weight 3.8-kb transcript or more likely from another as yet undiscovered member of the SST-28 selective SSTR subfamily.
为了解释AtT - 20细胞所特有的生长抑素- 28（SST - 28）结合选择性，对生长抑素受体（SSTR）基因和基因产物在AtT - 20细胞中的表达模式进行了表征。AtT - 20细胞表达多种SSTR mRNA。矛盾的是，这包括四种SST - 14选择性受体中的三种的mRNA：SSTR2（+）、SSTR1（+）、SSTR4（+）。与正常小鼠垂体相比，SST - 28选择性的SSTR5以相对低丰度（+）的3.8千碱基（kb）转录本形式表达，而正常小鼠垂体显示出高水平（ ）的2.4 kb SSTR5 mRNA。用抗肽SSTR2抗体对溶解的膜进行免疫印迹分析，发现了一种72±2 kDa的单一SSTR2蛋白。用SSTR2抗体对AtT - 20细胞膜进行预孵育，使125I - [Leu8，D - Trp22，Tyr25]SST - 28结合位点减少了38%。剩余的结合位点对SST - 28的亲和力增加了4.9倍，对SST - 14的亲和力降低了2.6倍，SST - 28:SST - 14效价比为40:1，而对照膜中的效价比为3.5:1。这些结果表明AtT - 20细胞中表达了四种SSTR基因，其中SSTR2占主导。用抗体阻断SSTR2会暴露出高亲和力的SST - 28选择性位点，其结合特性与报道的克隆SSTR5相当。这些SST - 28结合位点可能来自由高分子量3.8 kb转录本编码的SSTR5变体，或者更有可能来自SST - 28选择性SSTR亚家族中另一个尚未发现的成员。
