Chronic pain is experienced by 1 in 5 adults during their lifetime and is often poorly managed. Activating analgesic opioid receptors, for example, can provide short-term relief but often leads to unacceptable side-effects. As an alternative, blocking receptors such as the Neurokinin 1 Receptor (NK1R) - a GPCR that is known to transmit pain through the spinal cord - have long been pursued as a non-opioid analgesic targets, without clinical success. While this may be due to many reasons, our program has focussed on testing the hypothesis that when active, these receptors undergo endocytosis into endosomes and may therefore be ‘hidden’ from many therapeutics that bind in an untargeted manner. This talk will focus on our recent location-specific signalling studies, where we have employed a range of biophysical tools and imaging approaches to correlate Substance P-dependent NK1R signalling from internal membranes such as endosomes, with pain transmission in spinal neurons and in pre-clinical pain models. Furthermore, by developing lipid-peptide conjugates that can enhance the accumulation of peptide antagonists within specific membranes in cells, we demonstrate that targeted, location-specific peptide-receptor engagement in cells can enhance the efficacy of low potency, water-soluble peptide antagonists that would otherwise only inhibit cell surface GPCR signalling. These proof-of-concept studies suggest that in addition to improving peptide stability and PK profiles, peptide-lipid conjugates can also impart unique pharmacological effects that may be valuable for drug repurposing in pain and other chronic conditions.