Our work on “Proteins with multiple G protein-coupled receptor domains” is finally out on www.biorxiv.org/content/10.1101/2022.07.26.501653v1. Really excited about what we found – proteins that form “multi-GPCRs”, as outlined in the following 🧵(1/11)
(It's a 2GPCR predicted structure from a black rhino)

We found proteins that contain two, three, and even four entire #GPCR 7TM domains, stringed along one protein chain. This contrasts with the GPCRs with a single 7TM transmembrane domain that we all know and love (2/11)

#GPCRs are the most broadly distributed family of eukaryotic membrane receptors. Many form dimers, but all are from two individual protein chain, with the dimeric interactions often functionally meaningful. Now we see evolution also concatenated several GPCRs in one chain.(3/11)

We report 57 multi-GPCR proteins, identified with a sequence-based search, and then used #AlphaFold to predict their 3D structures. Here is an example: the predicted structure (with 2 7TMs) of A0A3L7HXX2from Cricetidae (a rodent)🐿️(pLDDT 81.5).(4/11)

These putative multi-GPCRs are found across Chordata, including in fish, birds, camels, marmites, and Chinese hamster. In primates, we found a few in new world monkeys(like Dusky titi monkey), but none in humans (we did look ;-) ). (5/11)

Individual #GPCRs 7TMs within multi-GPCRs have homologues (that's how we found them). Thus, #AlphaFold is confident about the 7TM predictions, but less regarding their relative positioning. Most genes are homologous to olfactory receptors, a few to chemokine receptors.(6/11)

We find the well-known #GPCR motifs DRY/NPxxY in almost all multi-GPCRs domains - a strong indication for signaling activities. But, we also found something curious🤔: linkers between the GPCRs are rather short and not very hydrophobic.(7/11)

#GPCRs function in a specific orientation in the membrane: the N-terminus on the outer-membrane side with each #GPCR TM helix crossing the membrane. After the 7-helices, the chain continues on the inner side of the membrane --\/\/\/\--. (8/11)

For two concatenated 7TMs to be in the same functional orientation, their linker should be long & hydrophobic. But even a 15-aa window in the linkers are not hydrophobic enough to traverse the membrane. This is confusing 🤔 but we offer three speculations for this puzzle.(9/11)

Maybe two-GPCRs are arranged in two opposite orientations but only one functions at any given time(A)? Maybe, a (yet to be identified) enzyme cuts the multi-GPCRs into individual ones (B)? Or maybe, the second GPCRs is in an internal membrane (C)? (10/11)

We find these multi-GPCRs very cool😎: revealing potential new complexity in signal transduction, with additional possibilities for signal regulation or amplification within the receptor. Go #GPCR evolution! With @KIsildayancan, @KesselAmit, Ron Solan, and Nir Ben-Tal (11/11)