Heterotrimeric G-proteins: a short history

Abstract

Some 865 genes in man encode G-protein-coupled receptors (GPCRs). The heterotrimeric guanine nucleotide-binding proteins (G-proteins) function to transduce signals from this vast panoply of receptors to effector systems including ion channels and enzymes that alter the rate of production, release or degradation of intracellular second messengers. However, it was not until the 1970s that the existence of such transducing proteins was even seriously suggested. Combinations of bacterial toxins that mediate their effects via covalent modification of the alpha-subunit of certain G-proteins and mutant cell lines that fail to generate cyclic AMP in response to agonists because they either fail to express or express a malfunctional G-protein allowed their identification and purification. Subsequent to initial cloning efforts, cloning by homology has defined the human G-proteins to derive from 35 genes, 16 encoding alpha-subunits, five beta and 14 gamma. All function as guanine nucleotide exchange on-off switches and are mechanistically similar to other proteins that are enzymic GTPases. Although not readily accepted initially, it is now well established that beta/gamma complexes mediate as least as many functions as the alpha-subunits. The generation of chimeras between different alpha-subunits defined the role of different sections of the primary/secondary sequence and crystal structures and cocrystals with interacting proteins have given detailed understanding of their molecular structure and basis of function. Finally, further modifications of such chimeras have generated a range of G-protein alpha-subunits with greater promiscuity to interact across GPCR classes and initiated the use of such modified G-proteins in drug discovery programmes.

Figure 1

Homology of mammalian G-protein α-subunits. The relatedness of individual mammalian G-protein α-subunits is shown as an unrooted homology tree. The date of cloning of cDNAs corresponding to each family member is shown in parentheses.

Figure 2

β/γ and receptor contact sites on Gα. Top: Sequence alignment of the N- and C- terminal regions of selected Gα-subunits. Residues that are subject to N-linked myristoylation, thio-palmitoylation or N-linked palmitoylation are highlighted in orange, green and yellow, respectively. In each case, M (black) is the protein synthesis initiator, methionine, that is eliminated during protein synthesis. Residues comprising the N-terminal αN helix are highlighted in red and residues at the extreme C-terminus of Gα are shown in blue. The αN helix is required for binding β/γ-subunits, and particular β/γ contacts are boxed in black, the extreme C-terminus plays a key role in specific receptor recognition. In the secondary structure diagram below the aligned sequences, β/γ and receptor interaction sites are highlighted in red and blue, respectively. Only selected domains of Gα are shown, and for simplicity the domains between αA and the α2 helix have been omitted as indicated by the dotted line. Bottom: Illustration of the N-terminal αN helix (red) and the C-terminal receptor contact region (blue) in the context of the tertiary and quaternary structure of the resting state, inactive G_i1αβ₁γ₂ heterotrimer. The GDP molecule is buried between the GTPase and helical domain of Gα (green), the β-subunit is coloured yellow and the γ-subunit is shown in orange. The diagram was generated using the coordinates from the PDB file 1GP2 and visualised with WebLab ViewerPro.

Figure 3

The nature of the G-protein α-subunit-bound nucleotide controls the extent and temporal kinetics of G-protein signaling. Conversion of a G-protein heterotrimer from the inactive, GDP-bound, to active GTP-bound state is promoted by interaction with a guanine nucleotide exchange factor (GEF), the most common of which are members of the GPCR family. Subsequent conformational changes promote separation of the GTP- bound α-subunit from the β/γ complex, whereupon both elements of the G-protein can regulate the activity of effector proteins that include second messenger generating enzymes and ion channels (see Table 1 for details). The intrinsic GTPase activity of the G-protein α-subunit hydrolyses the terminal phosphate of bound GTP and terminates function. This activity is accelerated by GTPase-activating proteins, the largest family of which are the regulators of G-protein signalling (RGS) proteins. Reassociation of Gα-GDP with the β/γ complex terminates effector regulation by the β/γ-subunits (Table 1) and completes the cycle. Further interaction with a GEF is now required to reinitiate the cycle.