Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


RUUSKANEN

Divergent 2-adrenoceptor subtypes in the zebrafish (Danio rerio)

Jori Ruuskanen1, Minna Varis2, Erik Salaneck4, Tiina Salminen2, Tommi Nyronen3, Mark S. Johnson2, Dan Larhammar4 and Mika Scheinin1

1Department of Pharmacology and Clinical Pharmacology, Univ of Turku, Finland
2Department of Biochemistry and Pharmacy, Akademi University, Turku, Finland
3Center for Scientific Computing (CSC), Espoo, Finland
4Department of Neuroscience, Unit of Pharmacology, Uppsala University, Sweden

2-Adrenergic receptors (2-AR:s) belong to a large family of G-protein coupled receptors. A common feature of these receptors is seven a-helical transmembrane domains (TM) thought to form the binding pocket for ligands. They mediate many of the physiological effects of adrenaline and noradrenaline and are target molecules for several drugs. Three human 2-AR subtype genes (2A, 2B and 2C) have been cloned to date. The number of 2-ARs in fish has remained unclear. Only one 2-AR in the fish Cuckoo wrasse (Labrus ossifagus) has been cloned. This receptor, named 2F, has been thought to represent an ancestral 2-AR subtype. Its ligand binding properties are intermediate between 2A and 2C. However, it shows greatest sequence similarity to the 2C and from an evolutionary point of view it is more likely that fish also have three 2-AR subtypes. To study the structure and evolution of 2-AR:s and their possible importance in developmental biology, we have turned to another species of fish, the zebrafish (Danio rerio), which is highly amenable for developmental and genetic studies.

We have cloned the genes coding for the zebrafish 2A-AR and 2C-AR. At protein level, both of these show around 60 % sequence identity when compared to their mammalian counterparts and 50 % identity when compared to other 2-AR subtypes. Analysis of the TM regions is based on a frog rhodopsin based model of the human 2A-AR. In the predicted TM regions, identity of the zebrafish 2A-AR with the human orthologue is 83.3 %, with the rat 2A 82.3 and with the mouse 82.3%. For the zebrafish 2C identities are: human 2C 80.3 %, rat and mouse 2C 86.9 %. These values are much lower than the percentage identities between human and rat or mouse orthologues; 97.5 % for the 2A and 98.5 for the 2C. The rat and mouse orthologues are 99-100 % identical. The greater diversity of the zebrafish receptors is expected to reveal certain residues important for a typical 2-adrenergic ligand binding, which in turn could help in designing subtype selective 2-drugs. Screening for additional subtype(s) using a probe corresponding to an 2B-AR like unpublished EST (GenBank acc. no. AI461341) has been carried out and further analysis of the resulting clones is in progress. Chromosomal mapping of the cloned receptor genes has been started in collaboration with Prof. John H. Postlethwait's group at the University of Oregon, Eugene, USA. Expression and pharmacological testing of the cloned receptor genes has also been started.


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