GPCR: G protein complexes--the fundamental signaling assembly

Abstract

G protein coupled receptors (GPCR) constitute the largest group of cell surface receptors that transmit various signals across biological membranes through the binding and activation of heterotrimeric G proteins, which amplify the signal and activate downstream effectors leading to the biological responses. Thus, the first critical step in this signaling cascade is the interaction between receptor and its cognate G protein. Understanding this critical event at the molecular level is of high importance because abnormal function of GPCRs is associated with many diseases. Thus, these receptors are targets for drug development.

Fig. 1

Rhodopsin signaling. Light activation of rhodopsin enables binding of its cognate G protein, transducin and triggers GDP→GTP exchange in its α subunit. Transiently, high affinity, nucleotide free rhodopsin-G_t complex is formed. Binding of GTP causes complex dissociation as well as dissociation of G_tα subunit from G_tβγ dimer and they activate different downstream pathways leading in effect to neuronal response in the brain. Dissociation of transducin from activated rhodopsin enables dissociation of is chromophore all-trans-retinal. Accumulated free opsin has to be regenerated with 11-cis-retinal in order to restore its function.

Fig. 2

Structural comparison of different photo-states of rhodopsin. a, Structures of ground state rhodopsin (1u19) (Pescitelli et al. 2008); b, photoactivated rhodopsin (2i35) (Salom et al. 2006); c, opsin (3cap) (Park et al. 2008); d, opsin with bound C-terminal peptide of G_t (3dqb) (Scheerer et al. 2008) and e, Meta II (3pxo) (Choe et al. 2011) are shown in dark red, salmon, blue, purple and green, respectively. f, All activated rhodopsin states were superposed on its ground state structure. Superposition of these structures show rearrangement of the cytoplasmic loops and movement of helix TM VI depicted with black ovals.

Fig. 3

Proposed location of GPCR-G protein complexes in a phospholipid bilayer. Semi-empirical model of the complex formed between light-activated rhodopsin dimer and a G_t heterotrimer (a) (Jastrzebska et al. 2011) and a structure of T4L-β₂AR-Gs complex (b) (Rasmussen et al. 2011b) were fitted into a 3D molecular map derived from EM-single particle reconstructions of negatively stained, crosslinked rhodopsin-G_t complexes purified in LMNG. Best fitting of the rhodopsin-G_t model was achieved for rhodopsin dimer and G_t hereotrimer (GOT1) after 30° hinge-like motion of the α-helical domain of G_t was applied. Fitting the T4L-β₂AR-Gs-nanobody structure into our EM 3D map leaves enough unoccupied space to accommodate a second molecule of this receptor, conformation of G_sα helical domain is inconsistent with our EM-density. The photoactivated rhodopsin that binds C-terminus of G_tα and molecule of T4L-β₂AR is depicted with yellow. The second rhodopsin molecule is shown in dark grey. Gα is colored pink; Gβ and Gγ are colored light grey.

Fig. 4

Asymmetry of activated rhodopsin (Rho*) dimer within rhodopsin-G_t (Rho*-G_t) complex. G_t binds to fully light activated rhodopsin (Rho) forming the Rho*-G_t complex. After light illumination, 11-cis-retinal (11-cis-RAL) of ground state rhodopsin (shown as red-red dimer) isomerizes to all-trans-retinal (all-trans-RAL) (yellow-yellow dimer) allowing binding of the G_t heterotrimer (G_tα is depicted in salmon, G_tβ in green and G_tγ in light blue). This binding prevents chromophore release from one rhodopsin in the dimer, introducing dimer asymmetry in the Rho*-G_t complex (shown as a yellow-grey dimer bound to G_t and black trace on the chromatogram₎. a, retinal-free rhodopsin molecule of the Rho*-G_t complex can be regenerated with 9-cis-retinal (9-cis-RAL) (shown as yellow-pink rhodopsin dimer bound to G_t and blue trays on the chromatogram). Release of chromophore from the G_t-protected rhodopsin molecule can be promoted by NH₂OH, resulting then in formation of an asymmetric retinal free and 9-cis-retinal regenerated rhodopsin dimer complexed to G_t (shown as grey-pink rhodopsin dimer bound to G_t and dark cyan trace on the chromatogram). Regeneration of this complex with 11-cis-retinal surprisingly results in formation of all-trans-retinal bound and 9-cis-retinal bound asymmetric rhodopsin dimer in the complex with G_t (shown as yellow-pink rhodopsin dimer bound to G_t and cyan trace on the chromatogram). b, the chromophore-free Rho*-G_t complex (modeled as a grey-grey rhodopsin dimer bound to G_t and black lane on the chromatogram) can be generated by treatment of the Rho*-G_t complex with NH₂OH. Then this chromophore-free Rho*-G_t complex can be regenerated with 9-cis-retinal which binds to both rhodopsin monomers within the dimer (shown as a pink-pink dimer bound to G_t and dark green trace on the chromatogram). Alternatively, it can be regenerated with all-trans-retinal but only one rhodopsin molecule, the one directly coupled to G_t binds all-trans-retinal (shown as a yellow-grey dimer bound to G_t and light green trace on the chromatogram).