GAIN, subdomain B (GAIN_B)
Type: Domain

This entry represents the entire subdomain B of GAIN.

The ~320-residue GAIN domain is an ancient domain that exists in primitive ancestor organisms, and is conserved in all aGPCRs and all PKD1-related proteins. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine-tunes the chemical environment. In contrast to most other autoproteolytic domains that cause the release of the cleaved mature protein, autoproteolysis of the GAIN domain does not lead to the dissociation of the cleaved extracellular sequences containing the GAIN domain from the membrane-embedded regions of the protein. The GAIN domain forms a tightly associated heterodimer upon proteolysis. Cleavage at the GAIN domain is not a feature that all GAIN domain-containing receptors possess and the GAIN domain likely has other functions (such as interacting with ligands or transmembrane helices) in addition to the autoproteolysis function.

The conservation of the primary sequence of the GAIN domain decreases from the C terminus to the N terminus, and the GPS motif is the most conserved region of the domain. The N-terminal subdomain A of the GAIN domain is composed of a variable number of α-helices (6, 5 or 3), while the C-terminal subdomain B consists of a twisted β-sandwich including 13 β-strands and 2 small α-helices. The autocatalytic scissile bond in the GPS of the GAIN domain is positioned at a sharply kinked loop between the last two β-strands of the GAIN domain and lies close to the core of the protein away from the surface. Autoproteolysis cleaves the C-terminal β-strand from the rest of the domain. The C-terminal β-strand of the GAIN domain corresponds to the "Stachel peptide" (also called "tethered agonist" or "stalk") and acts as the tethered agonist that activates the aGPCR. However, autoproteolysis does not cause the dissociation of the Stachel peptide from the rest of the GAIN domain, and it is unclear how it can be exposed to the transmembrane region to activate the receptor. The Stachel peptide is highly conserved and has a hydrophobic nature. It is suggested that mechanical force applied on the extracellular domains may lead to shedding of the extracellular regions and release of the Stachel peptide from the GAIN domain enabling the Stachel peptide to activate the receptor .

The proposed autoproteolytic mechanism suggests that a general base at position -2 respective to the cleavage site (such as a histidine in rat
latrophilin 1 or a water molecule) retracts a proton from the hydroxyl group of a threonine/serine at position +1 to yield a negatively charged oxygen.
This oxygen makes a nucleophilic attack on the carbonyl carbon of the residue at position -1 (a leucine in rat latrophilin 1) forming a tetrahedral
intermediate followed by an ester intermediate that eventually produces the cleaved protein. In most GAIN domains, the cleavage site is HL|T/S/C. Although autoproteolysis can occur only if the residue at position +1 is a threonine,serine, or cysteine, the residues at positions -2 and -1 may vary in different GAIN domains .

The GPS motif is an integral part, and the most conserved region, of a much larger (320-residue approximately) domain that has been termed GPCR-Autoproteolysis INducing (GAIN) domain. The GAIN domain represents an autoproteolytic fold whose function is relevant for GPCR signalling and may regulate this process . The GPS motif is an essential element for receptor function and needs to be within the context of the GAIN domain to mediate autoproteolysis .