ABSTRACT Adhesion G-Protein Coupled-Receptors (aGPCR) represent a particularly interesting class of receptors in the understudied druggable genome that could be therapeutic targets for pain control. These receptors possess tethered peptide agonists on the N-terminus of the proteins that are exposed by autoproteolysis (Stachel sequence). After proteolysis the receptor remains active until it is degraded, which could chronically maintain pain. The Eligible Protein List contains 27 aGPCRs. In our recent transcriptomic analysis of mouse trigeminal ganglia (TG) we found that 23 of these proteins are expressed in TG, yet only ADGRD1 (UniProt# Q6QNK2) was reported to be altered by infraorbital nerve chronic constriction injury (ION-CCI). ADGRD1’s Stachel sequence is highly selective for the receptor suggesting that the binding site is discriminative and that selective ligands could be designed to interact with the protein, the receptor activates Gαs to stimulate cAMP production, and the receptor is expressed at similar levels in mouse, rat, and human TG indicating that it should be translatable. These data suggest that ADGRD1 maybe an exceptional candidate for analgesic development. However, to date ADGRD1 has not been examined as a potential mediator of pain. This project will address the following questions to establish the function of ADGRD1 in ION-CCI induced chronic allodynia. 1) What sensory neurons express ADGRD1? This aim will utilize transcriptomics and immunohistochemistry to identify the cell types in the TG and trigeminal subnucleus caudalis (TNC) that express ADGRD1 in naïve and ION-CCI mice. These experiments will also evaluate the relationship between the development of ION-CCI mechanical allodynia and protein expression. 2) Can ADGRD1 be activated by its Stachel peptide when applied exogenously? The presence of the receptor in TG in naïve mice suggests that administering the Stachel peptide to the animals could induce facial mechanical allodynia, which would confirm a role for the protein in nociception. 3) Can ION- CCI induced mechanical allodynia be suppressed by a targeted botulinum toxin light chain that uses the Stachel peptide as a targeting sequence? This aim will take advantage of our previous experience in the development of neuropeptide – toxin conjugates to either lesion or alter the function of nociceptive neurons. A botulinum A light chain conjugate with the ADGRD1 Stachel peptide (Stach-Bot) will be constructed and tested for its ability to block ION-CCI induced mechanical allodynia in mice. These experiments would confirm that the neurons expressing ADGRD1 are nociceptive and that they participate in mechanical allodynia. The data collected in this project will establish the cell types that express ADGRD1, determine if the protein can produce allodynia when activated, and demonstrate that the neurons that express ADGRD1 participate in ION- CCI induced allodynia. These results would provide the impetus for future studies t...