Analyzing Functional Divergence Of The Dopamine Receptors In Vertebrates

Objective:Dopamine is the important neurotransmitter in the central nervous system of the vertebrate and regulates a wide variety of functions via its binding with dopamine receptors.Abnormalities in dopamine receptors have also been proved to be related to various nervous system diseases such as Parkinson's disease,schizophrenia and Alzheimer's disease etc.Therefore,the study of the genes related to dopamine provides the important theoretical direction for the research and treatment of various diseases.So it is important to study the evolition of the genes related to dopamine and the functional divergence of the dopamine receptors,however there are no researchs related to the functional divergence of the dopamine receptors.In this study,we collected the cDNA and amino acid sequences of five dopamine receptor genes,and conducted a phylogenetic analysis of dopamine receptor genes in vertebrates to investigate their molecular evolution features.We also analyzed functional divergence in the dopamine receptors and the functional divergence sites.Methods:1.The cDNA and protein sequences of five dopamine receptor genes of ten vertebrates were collected,i.e.,Homo sapiens,Pan troglodytes,Macaca mulatta,Canis lupus familiaris,Bos taurus,Mus musculus,Rattus norvegicus,Gallus gallus,Xenopus tropicalis and Danio rerio.2.In order to detect the evolutionary rate of five dopamine receptor genes,we calculated d N/dS values for receptor genes using CODEML of PAML.3.We conducted those collected sequences by deleting the signal peptides.Then we conducted multiple alignments and phylogenetic analysis of dopamine receptor genes.4.For the dopamine receptor genes,we calculated the the functional divergence coefficients on the basis of multiple sequence alignment and phylogenetic analysis.5.We conducted a sites-special profile to infer the functional divergence related sites and analyzed the relationship between structure and function of dopamine receptor by combining with the protein structure.Results:1.Totally,we collected 47 cDNA and 47 amino acid sequences of 5 dopamine receptor genes,i.e.,D1-D5.2.For each dopamine receptor genes,dN/dS ratios are much smaller than 1,indicating a lack of positive selection in the evolution.In the DRD1-like dopamine receptor family,DRD1 evolved slower than DRD5.DRD2 had the lowest evolutionary rate among three DRD2-like subtype dopamine receptors.3.The phylogenetic tree showed the evolutional relation among different receptors.DRD2,DRD3 and DRD4 formed one cluster,which belonged to the D2-like type.The subunits DRD1 and DRD5 formed one cluster,which belonged to the D1-like type.Since receptors of the same type were more similar in function,the topology of phylogenetic tree and the functional classification were coincident.4.Our results showed that type I functional divergence,i.e.,altered functional constraints or different evolutionary rate,may have taken place at some amino acids in the receptors.There were two divergence events among dopamine receptors subunits.The divergence I happened between D1-like type and D2-like type.The divergence II happened among D2-like type subunits and D1-like subunits respectively.5.Under the condition(Qk>cut-off value),we got 84 different sites.These sites are not evenly distributed in the sequences,most of these are located in the 384-578 of the alignment sequence.When these sites were mapped to the 3D structure of dopamine receptors,most of them were included in ICL3,M6 and M7 domains.These sites can be molecular basis for further study.Conclusion:1.In the analysis of functional divergence,there were two divergence events among dopamine receptors subunits.The divergence I diverged D1-like type and D2-like type subunits.The divergence II happened among D2-like type subunits and D1-like subunits respectively.Therefore,a stsble difference is formed between the D1-like type and D2-like type subunits after gene replication.2.There are 84 sites related to type I functional divergence for the pairwise comparisons between the D1-like and D2-like are identified in total,most of them were included in ICL3,M6 and M7 domains.Especially,the 16 sites may be the major sites associated with properties change between D1-like and D2-like receptors.3.However,there were also sites that were related to functional divergence both between and within the receptor families,e.g.,D177,P178,T179,N303 and G304.These sites can be molecular basis for further study.