Combinatorial GPCRs.

A recent paper published in the journal EMBO- on the structural dynamics of G protein-coupled receptor (GPCR) oligomerization- is particularly interesting, as it relates to a paradigm that wasn’t in play until very recent. This class of cell-surface receptors comprises the largest known protein superfamily, and its members are characterized by their signature heptahelical membrane-spanning domains. These seven transmembrane receptors are implicated in a myriad of stimulus-response pathways, and have served as incredibly lucrative drug targets- roughly 40-50% of all modern pharmaceuticals target GPCRs.

A general signal transduction pathway can be described. Receptors are activated by extracellular ligands that bind to the extracellular interface, helping shift receptor equilibrium towards the active state conformation. There is great diversity in the set of ligands, which includes photons, peptides, biogenic amines, lipids, and most recently demonstrated, voltage across a membrane. On the intracellular side of the cell membrane, receptors are associated with G proteins (heterotrimeric guanine nucleotide-binding proteins) composed of a GDP-bound alpha subunit and a beta-gamma dimer. Receptor activation promotes G protein activation: the alpha subunit swaps GDP for GTP, followed by dimer disassociation. This activated GTP-bound alpha subunit can then act on a given effector, which in turn initiates a second messenger signalling cascade. The specific effector and the mode of action is dictated by the subtype of the alpha subunit that couples to a given receptor.

Now for a little history. Until 1999, it was assumed that GPCRs behave exclusively as monomers, with little evidence to the contrary. It was then shown that GABA type B R1 and R2 receptors (two divergent GPCR-encoding genes) form a hetero-dimer as a requisite for receptor functionality (Marshall et al, 1999). It wasn’t until 2000 that a similar phenomenon was established and reported in class I (A) GPCRs. The hetero-dimeric association of SST5 (a serotonin receptor) and D2 (a dopamine receptor) further complicated the picture by revealing that distantly related receptors may co-evolve to form higher order structures (Rocheville et al, 2000).

“The GPCR family is probably the largest in the human genome. If ligand-induced heterodimerization turns out to be common, then the array of GPCR combinations will be truly bewildering.” -Graeme Milligan (Receptors as Kissing Cousins, Perspectives)- [Science 7 April 2000].

This underscores the importance of these findings. These receptors (as a whole) could no longer be thought to necessarily or exclusively display ligand sensitivity as monomeric units. Examples of hetero and homo-dimers (and higher-order oligomers) have poured in over the last few years. This adds huge complication to the elucidation of receptor structure and function, their ligands, their downstream biochemical effectors, and their further downstream physiological consequences. As if the shear number of receptors wasn’t challenging enough to deal with, now the idea that they can combinatorially form superstructures must also be factored into their characterization, and mapping a ligand-receptor-G protein network becomes a much more computationally difficult problem.

The paper that sparked this entry, “Ligand sensitivity in dimeric associations of the serotonin 5HT2c receptor” (Mancia et al), helps further resolve dimer structure, more specifically, the conformational dynamics of a quasi-symmetric homo-dimer in various states of activation. The actual receptor placed under an experimental lense is a Gq-coupled class I 5HT (serotonin) receptor, involved in anxiety and depression, and differentially expressed in the choroid plexus (it plays some role in CSF release). The physiology, as always, is less than interesting.

The authors employ a cysteine cross-linking approach to “capture” the 5HT homo-dimer, as it exists and behaves natively. Residues were selected for site-directed mutagenesis to maximize the probability of cross-link formation, based on mapping this receptor’s primary structure to the only available x-ray crystallography structure of a Class I GPCR (Rhodopsin). The homologous Rhodopsin residue was to be 6-8 Å from the cell surface, and sufficiently exposed on the receptor surface (>40% surface area). In total, 46 sites were selected (30 original sites, in addition to 16 near amino acids), and all transmembrane domains were targeted.

Expression vectors were constructed for each mutant, and transiently transfected into the HEK293 cell line. There were 45 individual transfections, and 323 combinatorial transfections. Co-transfection pairs were selected such that the calculated distance between mutated residues would fall within 5 Å. All transfected lines were first exposed to the hyperoxidizing environment induced by Cu-P (Cu-Phrenanthroline). Of the original batch, and as resolved by western blotting, 30 transfections exhibited interactions (a band at roughly 90 kDa corresponding to dimer formation via cysteine cross-link). The experiment was repeated on this smaller subset, with a much more stringent oxidizing environment. A single interface was discovered at TM I-I, and two arose at TM IV-V.

The researchers then went on to test the efficacy of known ligands on the three homo-dimers, in the hopes of identifying at least one exhibiting the proper ligand sensitivity profile. Only one of the original TM IV-V co-transfections (I193C/P213C) survived, displaying a quantifiable increase in receptor activation as they moved from inverse agonist, to absence of ligand, to antagonist, to agonist. More importantly, one particular pair of co-transfected mutants led to the mimicking of the functional profile of an authentic receptor dimer, while shifting a single amino acid on the TM V domain (I193C/N214C) abolished this sensitivity. Their results were clear indication that a substantial conformational change occurs at the TM IV/V interface during 5HT2c receptor activation.

Finally, the nature of dimer symmetry was explored. Do these receptors form dimers in a two-fold symmetry fashion, where IV-V’ and IV’-V interactions are identical? Or are the dimers quasi-symmetric? A switch to double-mutant transfections helped solve this problem, by helping show that both single and dual 193/214 links form comfortably, while dual 193/213 links destabilize the receptor. The next experiment focused on the critical 193/213 pairing, and alternatively fused Gαq C-terminal fusions were constructed. The hypothesis was that G protein location shouldn’t matter for a symmetrical homodimer, but one alternative would likely be preferable for a conformationally asymmetrical dimer. I193C/P213C-Gαq was preferred over I193C-Gαq/P213C double mutants, consistent with Gα stabilization in an asymmetrical dimer.

The take home message is that this homo-dimer is essentially a conformational hetero-dimer. They end by speculating and providing a pretty structural depiction that is likely to extend to other such class I GPCR oligomers (and beyond).