Class A G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors in the human genome and are involved in the regulation of many physiological functions. Understanding the mechanisms that drove the evolution of this receptor family should allow a better knowledge of sequence-structure-function relationships of the different sub-families. To gain evolutionary information on GPCRs, we explored their sequence space by metric multidimensional scaling (MDS). We applied a new MDS technique which projects supplementary sequences onto a sequence space of reference and allows comparison of sequences from different species. Results show that receptors cluster in four groups and suggest that modern receptors evolved from ancestors of the peptide receptors along three main evolutionary pathways. Proline residues of transmembrane helices 2 and/or 5 are involved in two of these pathways. To further detail the mechanisms that led to the different sub-families, we analyzed covariations of residues at different hierarchical levels (class/group/sub-family). We tested different methods of covariation analysis in order to select a method robust at the different hierarchical levels. This method highlights sequence determinants that are crucial for the evolution of specific sub-families.
