Natural G-protein-coupled receptors (GPCRs) rarely have an additional transmembrane (TM) helix, such as an artificial TM-linker that can unite two class A GPCRs in tandem as a single-polypeptide chain (sc). Here, we report that three groups of TM-linkers exist in the intervening regions of natural GPCR fusions from vertebrates: (1) the original consensus (i.e., consensus 1) and consensus 2~4 (related to GPCR itself or its receptor-interacting proteins); (2) the consensus but GPCR-unrelated ones, 1~7; and (3) the inability to apply 1/2 that show no similarity to any other proteins. In silico analyses indicated that all natural GPCR fusions from Amphibia lack a TM-linker, and reptiles have no GPCR fusions; moreover, in either the GPCR-GPCR fusion or fusion protein of (GPCR monomer) and non-GPCR proteins from vertebrates, excluding tetrapods, i.e., so-called fishes, TM-linkers differ from previously reported mammalian and are avian sequences and are classified as Groups 2 and 3. Thus, previously reported TM-linkers were arranged: Consensus 1 is [T(I/A/P)(A/S)–(L/N)(I/W/L)(I/A/V)GL(L/G)(A/T)(S/L/G)(I/L)] first identified in invertebrate sea anemone Exaiptasia diaphana (LOC110241027) and (330-SPSFLCI–L–SLL-340) identified in a tropical bird Opisthocomus hoazin protein LOC104327099 (XP_009930279.1); GPCR-related consensus 2~4 are, respectively, (371-prlilyavfc fgtatg-386) in the desert woodrat Neotoma lepida A6R68_19462 (OBS78147.1), (363-lsipfcll yiaallgnfi llfvi-385) in Gavia stellate (red-throated loon) LOC104264164 (XP_009819412.1), and (479-ti vvvymivcvi glvgnflvmy viir-504) in a snailfish GPCR (TNN80062.1); In Mammals Neotoma lepida, Aves Erythrura gouldiae, and fishes protein (respectively, OBS83645.1, RLW13346.1 and KPP79779.1), the TM-linkers are Group 2. Here, we categorized, for the first time, natural TM-linkers as rare evolutionary events among all vertebrates.

天然 G 蛋白偶联受体 (GPCR) 很少有额外的跨膜 (TM) 螺旋，例如可以将两个 A 类 GPCR 串联起来作为单多肽链 (sc) 的人工 TM 连接体。在这里，我们报道了脊椎动物天然GPCR融合的介入区域中存在三组TM连接子：（1）原始共有序列（即共有序列1）和共有序列2~4（与GPCR本身或其受体相互作用有关）蛋白质）； (2) 共识但与GPCR无关的，1~7； (3) 无法应用与任何其他蛋白质没有相似性的 1/2。计算机分析表明，所有来自两栖类的天然 GPCR 融合体都缺乏 TM 连接子，而爬行动物则没有 GPCR 融合体；此外，在来自脊椎动物（不包括四足动物，即所谓的鱼类）的（GPCR单体）和非GPCR蛋白的GPCR-GPCR融合物或融合蛋白中，TM连接子不同于先前报道的哺乳动物并且是鸟类序列并被分类为作为第 2 组和第 3 组。因此，先前报道的 TM 连接子被排列：共识 1 是 [T(I/A/P)(A/S)–(L/N)(I/W/L)(I/A /V)GL(L/G)(A/T)(S/L/G)(I/L)] 首次在无脊椎动物海葵Exaiptasia diaphana (LOC110241027) 和 (330-SPSFLCI–L–SLL-340)中发现在热带鸟类Opisthocomus hoazin蛋白 LOC104327099 (XP_009930279.1) 中鉴定出； GPCR相关共识2~4分别为沙漠林鼠Neotoma lepida A6R68_19462 (OBS78147.1)中的(371-prlilyavfc fgtatg-386)、Gavia星状潜鸟(红喉潜鸟)中的(363-lsipfcll yiaallgnfi llfvi-385)蜗牛 GPCR (TNN80062.1) 中的 LOC104264164 (XP_009819412.1) 和 (479-ti vvvymivcvi glvgnflvmy viir-504)；在哺乳动物Neotoma lepida、Aves Erythrura gouldiae和鱼类蛋白（分别为 OBS83645.1、RLW13346.1 和 KPP79779.1）中，TM 连接子属于第 2 组。在这里，我们首次对天然 TM 连接子进行了分类作为所有脊椎动物中罕见的进化事件。