BMC Genomics 2009, 10(Suppl 1):S14 http://www.biomedcentral.com/1471-2164/10/S1/S14 to have happened at approximately the time of the emer- gence of early vertebrates and mammals [2]. Massive gene duplications would bring great disturbance to the gene regulatory networks in the cell. How gene duplications impacted and reshaped the gene networks was still not well understood. Nevertheless, several recent theoretical analyses have shed some light on the issue [5-8]. It was shown that the scale-free properties of the gene networks were necessary consequences under the assumption of asymmetric retention of duplicated genes in favor of the genes in the periphery of the network, which was sup- ported by the family sizes of genes with different connec- tivity in genetic or protein-protein interaction (PPI) networks in yeast and worm [5,7]. However, these studies did not provide information as to how the duplication-divergence process [5] proceeded along the time axis during major speciation events, such as the emergence of vertebrates, as their model species were all invertebrates. Meanwhile, the genetic or PPI net- works offered only snapshot information about the rela- tionship between family sizes and connectivity of genes, which was often found to be inaccurate. Independent evi- dences not directly based on genetic or PPI networks were needed for cross examination. In view of these problems, in this study, we used human G-protein coupled receptors (GPCRs) and their down- stream genes in the pathways ("downstream genes") as the model system to examine the impact of gene duplica- tion on the evolution of genes in different layers of the network. It has been shown that the gene regulatory net- work roughly maps to the cellular organization, with the genes on the periphery of the cell maps to the peripheral layer of the gene network [9]. In this sense, GPCRs and their "downstream genes" offered a natural partition of the peripheral layer and the backbone layer of the gene network. Meanwhile, GPCRs also form one of the largest known groups of signaling proteins in mammalian genomes [10], and GPCR pathways cover a good portion of the gene network and influence a wide range of physi- ological activities such as neurotransmission, metabo- lism, secretion, differentiation and growth, learning and memory, and immune responses [11-13]. The results from the GPCR model system were thus highly represent- ative of the general gene regulatory network in human cells. In this study, we estimated the ages of the duplication events in human GPCRs and the "downstream genes" gene families. Comparison of the age distributions of GPCRs vs. the "downstream genes" provided a more detailed view of the duplication-divergence process along the time axis in the context of major speciation events in vertebrates. Furthermore, GPCRs were partitioned accord- ing to the GRAFS system [14] into subfamilies, and the age distributions of major subfamilies of GPCRs were esti- mated and compared. We also examined the expression profiles of GPCRs and downstream genes of different duplication ages, for their contribution to the tissue com- plexity at different evolutionary stages. In general, we found that most of the GPCR pathways, which cover a substantial portion of the gene network, have been estab- lished at the time of early vertebrate emergence. Contin- ued expansions in GPCR families were to a large extent contributed by several small subfamilies involved in immune responses and sensory responses. Our study of the GPCR pathways suggested that the position of a gene in the gene network has great influence on the likelihood of fixation of its duplicates. However, the influence was not uniform. Instead, expansion of a large gene family may be attributed to strong expansions of some particular subfamilies, when it was favorable in particular species, or at particular evolutionary stages. The generality of these principles could be further examined in other super gene families when the data become available. Results Defining the model system of GPCRs and the "downstream genes" Our model system of human GPCRs and the "down- stream genes" was based on the classical GPCR signal pathways, which has been summarized in [15]. Typically, a stimulated GPCR activates one or more types of G pro- teins (the transducers) which further activate specific effectors of various downstream pathways. G Proteins are composed of an α-subunit and a βγ-subunit (heterodimer stable at physiological conditions). The specificity of the cellular downstream pathways of GPCRs is largely deter- mined by the type of Gα subunits it binds. There are four major subfamilies for Gα, including Gαs, Gαi/o, Gαq/11, and Gα12/13, which define the corresponding GPCR- related signaling pathways: Gs, Gi/o, Gq/11, G12/13 and Gβγ pathways. We included the major genes in these pathways from the summary [15] as the "downstream genes", including the G proteins but excluding any genes that were cross-talking receptors or ion-channels on the cell membrane. The "downstream genes" in the pathways were summarized in Figure 1. The Gβγ pathway was not specifically shown as all its downstream genes were cov- ered in the Gi/opathway already. Vertebrate and inverte- brate homologues of the human "downstream genes" were identified and added to the families in order to reconstruct the phylogenetic trees and estimate of the ages of the duplication events (see Methods). We studied the human non-olfactory GPCRs in this study which were classified based on the GRAFS classification system [14]. The olfactory GPCRs were not included in this study, as their evolutionary patterns in mammals was Page 2 of 10 (page number not for citation purposes)