(a) Structure of the dCIRL GPS region. The GPS separates NTF from CTF in proteolyzable aGPCRs. The C-terminal cleavage component contains the Stachel sequence, a potent receptor agonist in many aGPCRs (light blue). Magenta: conserved, mutated residues that are necessary for GPS cleavage. (b) Western blot of whole fly protein extracts containing wildtype or proteolysis-defective GPS variants of dCIRL probed against an mRFP tag in the NTF. The dCIRL-GPSwt sample displays only a fragment corresponding to the cleaved NTF (ca. 106 kDa; filled circle), while the two GPS mutants contain a band representing the full-length receptor (ca. 218 kDa; open circle). (c) SIM images of dCIRLN-RFP fusion proteins with wildtype and proteolysis-resistant GPS in lch5. The protein is trafficked into dendrites and cilia, regardless of autoproteolytic cleavage. Scale bar 5 µm. (d) Receptor current recordings (average of 8 sweeps) of lch5 neurons under TTX inhibition highlight the divergent effects of the GPS mutations on mechanosensitivity (dark blue, dCirlH>A; light blue, dCirlT>A). (e) Quantification of tonic and phasic receptor current components. Despite abrogating GPS cleavage, the response profile of the dCirlH>A receptor variant is unaffected (900 Hz, phasic: p=0.464, tonic: p=0.460, Student’s t-test vs. dCirlRescue). In contrast, changing the first residue of the Stachel sequence in dCirlT>A mutants abolishes the receptor’s mechanosensory function, resulting in a dCirlKO response profile (900 Hz, phasic: p=0.030, tonic: p=0.023, Student’s t-test vs. dCirlRescue). Data are presented as mean ± SEM, n = 8 larvae per genotype.