Liapakis G, Tallent M, Reisine T
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084, USA.
Metabolism. 1996 Aug;45(8 Suppl 1):12-3. doi: 10.1016/s0026-0495(96)90070-0.
Identification of the ligand binding domains of the somatostain (SRIF) receptors may facilitate the rational development of new SRIF ligands. To identify ligand-binding domains of sst1, and sst2, we tested a series of chimeras. Using site-directed mutagenesis, we found that to bind with high affinity to sst2, the sst2 agonists MK678 and SMS-201-995 require a four amino acid sequence (FDFV) at the border of the third extracellular loop and transmembrane 7. Transference of residue 294 in msst2 to sst1 conferred onto sst1 the ability to bind SMS-201-995 and other octapeptides. Cyclic peptides with a phenylalanine adjacent to the D-Trp appear to interact with Phe294 of sst2, whereas hexapeptides with a tyrosine adjacent to the D-Trp, such as MK 678 and BIM 23027, did not interact with the Phe294. We have recently identified a peptide that selectively binds to human (h)sst1, with 100-fold higher affinity than for the other cloned SRIF receptor subtypes. The second extracellular loop of sst1 is critical for this peptide to bind. This contrasts with the sites involved in binding of sst2 agonists and indicates that the two receptors have distinct ligand-binding domains. G proteins couple SRIF receptors to multiple cellular effector systems, including adenylyl cyclase and ionic conductance channels. A critical cellular action of SRIF is the inhibition of Ca2+ influx, which may be responsible for its blockade of hormone and neurotransmitter release. Various studies suggest that both sst2 and sst5 endogenously expressed in AtT-20 cells can couple to L-type Ca2+ channels; the coupling was pertussis toxin-sensitive. The coupling of sst2 to the Ca2+ channels was relatively resistant to desensitisation; 5 hours of pretreatment with MK 678 did not attenuate MK 678 inhibition of the Ca2+ current. In contrast, the sst5 receptors were desensitised by 1 hour of pretreatment with BIM 23052. Thus, the coupling of the two receptors to the Ca2+ channel could be differentially regulated. The SRIF receptor subtype coupling to the Ca2+ channel could also be distinguished by a unique antagonist, the peptide L362,855, which binds with high affinity to cloned sst5.
确定生长抑素（SRIF）受体的配体结合结构域可能有助于合理开发新型SRIF配体。为了确定sst1和sst2的配体结合结构域，我们测试了一系列嵌合体。利用定点诱变，我们发现，sst2激动剂MK678和SMS - 201 - 995要与sst2高亲和力结合，需要在第三个细胞外环与跨膜7交界处有一个四氨基酸序列（FDFV）。将msst2中的294位残基转移到sst1上，使sst1具备了结合SMS - 201 - 995和其他八肽的能力。与D - Trp相邻有苯丙氨酸的环肽似乎与sst2的Phe294相互作用，而与D - Trp相邻有酪氨酸的六肽，如MK 678和BIM 23027，则不与Phe294相互作用。我们最近鉴定出一种肽，它能选择性地与人（h）sst1结合，其亲和力比其他克隆的SRIF受体亚型高100倍。sst1的第二个细胞外环对该肽的结合至关重要。这与sst2激动剂结合位点不同，表明这两种受体具有不同的配体结合结构域。G蛋白将SRIF受体与多种细胞效应系统偶联，包括腺苷酸环化酶和离子传导通道。SRIF的一个关键细胞作用是抑制Ca2 +内流，这可能是其阻断激素和神经递质释放的原因。各种研究表明，AtT - 20细胞中内源性表达的sst2和sst5都能与L型Ca2 +通道偶联；这种偶联对百日咳毒素敏感。sst2与Ca2 +通道的偶联相对不易脱敏；用MK 678预处理5小时并未减弱MK 678对Ca2 +电流的抑制作用。相反，用BIM 23052预处理1小时可使sst5受体脱敏。因此，这两种受体与Ca2 +通道的偶联可能受到不同的调节。与Ca2 +通道偶联的SRIF受体亚型也可用一种独特的拮抗剂——肽L362,855来区分，它能与克隆的sst5高亲和力结合。
