Oral Presentation 11th Australian Peptide Conference 2015

G protein-coupled receptors at the plasma membrane control most biological processes and are a major drug target. Activated receptors traffic to endosomes, but whether endosomal receptors generate signals that underlie complex pathophysiological processes and are viable therapeutic targets is unexplored. We examined whether the substance P (SP) neurokinin 1 receptor (NK₁R) and calcitonin gene-related peptide (CGRP) receptor (CLR) in endosome generate sustained signals that underlie pain transmission.

We used bioluminescence resonance energy transfer (BRET) and super-resolution microscopy to assess receptor trafficking and assembly of signaling complexes in HEK cells. We studied the generation of second messengers in subcellular compartments using Förster resonance energy transfer (FRET) biosensors. SP decreased NK₁R and KRas (resident plasma membrane protein) BRET and increased NK₁R and Rab5a (early endosomal protein) BRET, consistent with receptor endocytosis. The dynamin inhibitor Dyngo4a, the clathrin inhibitor Pitstop2, and dominant negative dynamin K44E, but not control compounds, inhibited endocytosis. Endocytic inhibitors and the Gαq inhibitor UBO-CIQ blocked SP-induced activation of cytosolic cAMP, cytosolic protein kinase C, and nuclear extracellular signal regulated kinase (ERK), indicating a key role for NK₁R endocytosis and Gαq in the full complement of NK₁R signaling events. Super-resolution microscopy confirmed the colocalization of NK₁R and Gαq in early endosomes. Endocytosis was similarly important for compartmentalized signaling of CLR.

In mice and rats, we investigated the effects of intraplantar injection of capsaicin, an agonist of transient receptor potential vanilloid 1 channel that stimulates SP and CGRP release from primary sensory neurons in the dorsal horn of the spinal cord. We quantified endocytosis of the NK₁R in spinal neurons by immunofluorescence and confocal microscopy, and assessed mechanical hyperalgesia using von Frey filaments. We also investigated pain induced by intraplantar injections of complete Freund’s adjuvant (CFA) and formalin. We examined the effects of SP and CGRP on excitability of spinal neurons by patch clamp recording in slice preparations. Intraplantar injection of capsaicin, CFA and formalin evoked NK₁R and CLR endocytosis and activation of ERK in lamina I spinal neurons, and caused sustained hyperalgesia. Intrathecal injection of Dyngo4a, Pitstop2, and dynamin-1 siRNA, but not control compounds, inhibited NK₁R endocytosis, ERK signaling, and hyperalgesia, revealing a major role for NK₁R endocytosis in pain transmission. Transient stimulation of spinal neurons with SP and CGRP caused a rapid yet sustained excitability that was suppressed by Dyngo4a, confirming the importance of NK₁R endocytosis for neuronal excitation.

To determine whether endosomal GPCRs may be a target for therapy, we generated lipid-conjugated antagonists of NK₁R and CLR. Lipidated antagonists rapidly accumulated in endosomes containing internalized NK₁R and CLR, and effectively blocked endosomally-derived signals. When injected intrathecally to mice, these antagonists blocked nocifensive behavior. They also suppressed excitability of spinal neurons in slice preparations.

These studies reveal a critical role for endosomal signaling of G protein-coupled receptors in the complex pathophysiology of pain, and suggest the utility of endosomally-targeted antagonists.