Bruns C, Raulf F, Hoyer D, Schloos J, Lübbert H, Weckbecker G
Sandoz Pharma Ltd, Basel, Switzerland.
Metabolism. 1996 Aug;45(8 Suppl 1):17-20. doi: 10.1016/s0026-0495(96)90072-4.
In the past few years, five different somatostatin (SRIF) receptor subtypes (sst1.5) have been identified, which form a distinct group in the superfamily of G-protein-coupled receptors. The naturally occurring somatostatins SRIF-28, SRIF-25, and SRIF-14 all reveal high-affinity binding for sst1.5. In contrast, short synthetic analogs that are in clinical use, such as SMS 201-995, RC-160, or BIM 23014, primarily interact with the sst2 subtype. Some SRIF analogs were previously reported to be selective for one SRIF receptor subtype, eg, the sst2 (MK 678), the sst3 (BIM 23056), or the sst5 (BIM 23052, L362-855) subtype. However, when we studied the binding affinities of these SRIF analogs for human (h) sst1.5 expressed in either CHO or COS-1 cells, we were unable to confirm these previously reported selectivities. The absence of sst antagonists is a major drawback for investigating the functional role of each sst subtype. We used site-directed mutagenesis to identify amino acids that determine ligand specificity for sst2. A single Ser305 to Phe mutation in TM VII increased the affinity of hsst1, for SMS 201-995 nearly 100-fold, and when Gln291 was also exchanged to Asn in TM VII of hsst1, almost full sst2-like binding of SMS 201-995 was obtained. These data may aid in the design and synthesis of new selective type sst ligands. We have identified the expression of sst subtypes in nonclassical SRIF target tissue such as the lung. The pKi values for SRIF and various SRIF analogs in rat lung tissue preparations were in close correlation with those obtained for CHO cells expressing the sst4 subtype. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the predominant expression of mRNA specific for sst4 in mouse, rat, and human lung tissue, confirmed by autoradiographies of rat lung. No specific binding for [125I]Tyr3-SMS 201-995 was detected, since SMS 201-995 has low affinity for sst4. In contrast, specific binding of [125I]SRIF-28 to rat lung sections was demonstrated, which could be displaced by unlabelled SRIF-14 and SRIF-28, indicating specific, high affinity binding of this radioligand to sst4 receptors.
在过去几年中，已鉴定出五种不同的生长抑素（SRIF）受体亚型（sst1 - 5），它们在G蛋白偶联受体超家族中形成一个独特的群体。天然存在的生长抑素SRIF - 28、SRIF - 25和SRIF - 14对sst1 - 5均显示出高亲和力结合。相比之下，临床使用的短合成类似物，如SMS 201 - 995、RC - 160或BIM 23014，主要与sst2亚型相互作用。一些SRIF类似物先前被报道对一种SRIF受体亚型具有选择性，例如sst2（MK 678）、sst3（BIM 23056）或sst5（BIM 23052、L362 - 855）亚型。然而，当我们研究这些SRIF类似物对在CHO或COS - 1细胞中表达的人（h）sst1 - 5的结合亲和力时，我们无法证实这些先前报道的选择性。缺乏sst拮抗剂是研究每个sst亚型功能作用的一个主要缺点。我们使用定点诱变来鉴定决定sst2配体特异性的氨基酸。在跨膜区VII中单个丝氨酸305突变为苯丙氨酸使hsst1对SMS 201 - 995的亲和力增加了近100倍，并且当在hsst1的跨膜区VII中谷氨酰胺291也被替换为天冬酰胺时，获得了几乎完全类似sst2的SMS 201 - 995结合。这些数据可能有助于新的选择性sst配体的设计和合成。我们已经确定了sst亚型在非经典SRIF靶组织如肺中的表达。大鼠肺组织制剂中SRIF和各种SRIF类似物的pKi值与在表达sst4亚型的CHO细胞中获得的值密切相关。此外，逆转录聚合酶链反应（RT - PCR）实验揭示了sst4特异性mRNA在小鼠、大鼠和人肺组织中的主要表达，大鼠肺的放射自显影证实了这一点。未检测到[125I]Tyr3 - SMS 201 - 995的特异性结合，因为SMS 201 - 995对sst4的亲和力较低。相比之下，证明了[125I]SRIF - 28与大鼠肺切片的特异性结合，其可被未标记的SRIF - 14和SRIF - 28取代，表明该放射性配体与sst4受体的特异性、高亲和力结合。
