Future phylogenetics analysis, including more extensive taxonomic sampling of unicellular relatives of Metazoa, invertebrates and primitive vertebrates will provide a more detailed understanding of evolutionary events that shaped the GRK family, including the picture of gene duplications and losses and, perhaps, of unequal evolutionary rates within the family (note many long branches in the GRK4/5/6 and GRK1/7 clades in ). Nevertheless, several conclusions about the GRK family evolution can be made with considerable degree of certainty. Most importantly, the RH+KD domain fusion is present in all animals, in two non-metazoan opisthokonts, and in three other lineages of the unicellular eukaryotes. The GRK kinase domain is most closely related to the kinases of the ribosomal protein S6 kinase family (
), which is ubiquitous in unicellular eukaryotes. Thus, it is most likely that the KD region of GRKs has been produced by duplication of the S6 kinase in the evolution of protists. The provenance of the RH domain is much harder to establish because of the fast evolution of this relatively short region and the uncertain placement of the RH domain of GRKs in the phylogenetic tree of RGS proteins 
, but it is notable that the RH domains are widespread in protists and should have been available for “domain tinkering”.
GRKs recognise and selectively bind activated GPCR, which act as allosteric activators. The N-terminus was implicated in kinase activation by active GPCRs. Mutations in this region specifically impaired the phosphorylation of GPCRs, but not other substrates 
. The N-terminus was proposed to interact with the kinase domain, stabilizing the alignment of the two lobes necessary for activity 
. The N-terminus was predicted to form α-helix 
, which was observed in the crystal structure of the most closed active-like conformation of any GRK solved thus far 
. Interestingly, in this structure the helical conformation of GRK6 N-terminus is stabilized by the crystal contacts, which were proposed to mimic GRK interaction with the receptor 
, support the proposed role of the N-terminal α-helix in GPCR-induced GRK activation. Therefore, it is likely to be the first element to acquire GRK-like characteristics. Our analysis demonstrates that N-terminal elements implicated in receptor binding are recognizable in GRKs from protists and are strictly conserved in all other species (). A stretch of positively charged residues between the starting N-terminal α-helix and the first helix of the RH domain characteristic of the GRKa/4/5/6 family was predicted to participate in GRK binding to the membrane and its proper orientation for receptor interaction 
. The elimination of these charges in GRK5 blocks the biding to PIP2
, and precludes PIP2
–dependent increase in receptor phosphorylation 
. The sequence comprising these positive charges () is remarkably conserved in all metazoan species from T. adherens
and cnidarians, and is also present in an elementary form in GRKa from a non-metazoan opisthokont Capsaspora owczarzaki
. Thus, it is most likely that this structural element evolved at the root of Metazoa to enhance the efficacy of GRK-mediated receptor phosphorylation.
A duplication of the ancestral GRK must have occurred at some point in the evolution before the emergence of Metazoa, to give rise to the lineage that includes GRKa/1/7/4/5/6 and the other, GRKb/2/3 lineage. Since two unicellular opisthokont genomes appear to encode one GRK each, and these GRKs may belong to two different clades, it is possible that GRKa and GRKb emerged in the opisthokont lineage and preceded the advent of multicellularity. Following this duplication, Monosiga may have lost GRKa, and Capsaspora lost GRKb. It is also possible that one or both of the “missing” genes simply remain un-annotated in the draft genome assemblies. The pre-duplication stage of GRK evolution is also represented by single-copy GRKs of Phytophtora, Albugo and brown alga.
The GRKa clade later split into GRK1/7 and GRK4/5/6 lineages – most likely in the early chordates, since an apparently ancestral “visual” GRK is present in the urochordate C. intestinalis
. This explains the lack of GRK1/7 genes in invertebrates. However, no visual GRK is found in amphioxus, possibly due to a secondary gene loss. Further gene duplications produced GRKs 2 and 3 in the GRK2/3 clade, GRKs 4, 5, and 6 in the GRK4/5/6 clade, and GRK1 and 7 in the GRK1/7 clade. It has been proposed that vertebrates evolved through two rounds of whole-genome duplications (the “2R” hypothesis), first at the root of the vertebrate lineage and the second when jawless vertebrates brunched off 
. The duplication in the GRKa clade, splitting the family into the GRK1/7 and GRK4/5/6 lineages, coincides with the first duplication. Further increase in the number of GRK isoforms is likely a part of the second duplication at the root of jawed vertebrates, since teleost fishes possess full complement of GRK isoforms. Unfortunately, no fully sequenced genomes between amphioxus and teleost fishes are available for comparison. Our BLAST search in a partial genome sequence of a cartilaginous fish elephant shark (Callorhinchus milii)
, which has recently become available 
, produced no matches. Cartilaginous fishes represent an oldest living group of jawed vertebrates and are important for understanding vertebrate evolution.
A third full-genome duplication is believed to have occurred in teleost fish lineage 
. The results of this duplication are reflected in the presence of two GRK5 paralogs in zebrafish. One rapidly evolving paralog, GRK5C, implicated in Wnt signalling in zebrafish 
diverged significantly in the kinase domain and evolved a novel C-terminus lacking membrane recruitment motifs typical for the GRKa/4/5/6 family. A similar fast evolving GRK5 paralog is present in pufferfish. This is consistent with asymmetrically accelerated post-duplication evolution of one of the paralogs 
. Duplicates for GRK4 and 6 appear to have been lost, and so has GRK2, substituted, possibly, by a GRK3 paralog. Teleost fishes also retained two closely related paralogs of GRK1 and possibly GRK7.
Split of the ancient eukaryotic GRK into opisthokont-specific GRKa and GRKb appears to have been followed very closely by acquisition of two distinct C-terminal extensions, a PH domain in GRKb and alternative membrane-targeting sequences in GRKa. The large partitions in the GRK kinase domain family trees show close correlation with the identity of the C-terminal regions of GRKs that are involved in membrane binding (). All proteins in the GRKb/2/3 clade in the tree, including the basal GRKb from Monosiga, have C-terminal PH domains; all GRK1/7 proteins have either farnesylation or geranylgeranylation CAAX motifs; and all GRK4/5/6 proteins have a predicted C-terminal amphipathic helix in one form or another. The shape of the helix with its characteristic arrangement of hydrophobic and positively charged residues specific for each GRK isoform is strictly conserved in vertebrates. In invertebrates lacking multiple GRK isoforms in the GRK4/5/6 clade, such as Chordata species lancelet and sea squirt, nematode C. elegans, placozoan T. adherens, and opisthokont C. owczarzaki, the form of the helix is GRK4-like, suggesting that this might be the ancestral form preceding gene multiplication in this clade. Insect species have their own unique strictly conserved arrangement, similar to that in cnidarian Nematostella vectensis. Thus, different arrangements of the lipid binding residues in the helix appeared in evolution of the GRKa/4/5/6, but some later disappeared or were modified. Although the precise mode of membrane interaction of invertebrate GRKa is unknown, distinct conservation of amphipathic helix suggests a mechanism similar to that operating in mammals.
The GRK1/7 clade is characterized by the presence of a C-terminal prenylation CAAX motif mediating membrane attachment of the members of this GRK subfamily 
. Mammalian rhodopsin kinases (GRK1) possess well-conserved CAAS motif and are farnesylated 
, whereas cone kinases (GRK7) possessing CAAL motif and are geranylgeranylated 
. The type of the C-terminal motif correlates well with the placement based on KD sequence (), with one exception: Gallus gallus
rhodopsin kinase robustly placed in the GRK1 group has CGVL geranylgeranylation motif. The basic member of the “visual” group, a GRK from C. intestinalis
, also has a geranylgeranylation motif CALL. Thus, geranylgeranylation may have been the lipid modification in an ancestral GRK in the “visual” branch that later changed to farnesylation in the GRK1 subfamily.
The G protein-coupled receptor kinases appear to be a more recent and more restricted addition to the GPCR signaling pathways compared to GPCRs themselves, which are found in nearly all eukaryotes 
. The origin of some of the GRAFS (G
ecretin) GPCR families could be traced to common ancestor of Uniconts and Alveolates at the very root of eukaryote evolution 
. We have not found GRKs in plant, fungi, or amoebozoa, whereas all these groups possess GPCRs. It is possible that GRKs have not been properly annotated in these genomes or there was a secondary loss of GRKs in all these lineages except Metazoa and related unicellular groups. GRKs point of origin may be closer to the timing of heterotrimeric G proteins and arrestin-like proteins, which are also found in all Metazoa and in many unicellular eukaryotes. The complete system of phosphorylation recognition, which includes GRKs and arrestins that specifically bind phosphorylated receptors 
, must have appeared at least in the ancestral opisthokont lineage that led to Metazoa. Indeed, M. brevicolus
genome encodes an arrestin-like protein; C. owczarzaki
genome encodes two, whereas other GRK-containing unicellular Chromalveolate eukaryotes do not appear to encode any. However, GRKs from unicellular oomycetes and brown algae, although quite different from metazoan GRKs, have perfectly recognizable N-terminal receptor-binding motive (), which suggests that these GRKs might be able to bind and phosphorylate GPCRs. The development of the complete GPCR desensitization system that includes both GRK-dependent receptor phosphorylation and arrestin binding to phosphorylated GPCRs may be linked with the expansion of the Rhodopsin GPCR family that also evolved in the common ancestor of Opisthokonts and enjoyed a huge evolutionary success in Metazoan lineage 
. It is likely that both these molecular inventions were of use to emerging fast-moving multicellular animals that are constantly surveying the environment with sophisticated sensory systems. This lifestyle requires the ability to reset and re-engage the environmental sensors frequently, which calls for the rapid shut-off mechanism. A dedicated system for quick deactivation of GPCR may have been one of the factors profoundly determining the metazoan lifestyle.