AbstractThe cannabinoid type one receptor (CB1R) is one of the most abundant G-protein coupled receptors (GPCRs) in the central nervous system. CB1R activation at the presynaptic terminal by ligand released retrogradely from the postsynapse dampens down neurotransmitter release. Consistent with its presynaptic role, CB1R is only stably surface expressed in axons. How this tightly regulated axonal surface polarity is established and maintained is unclear.
To address this question, I applied retention using selective hooks (RUSH) to visualise the trafficking of CB1R from biosynthesis to mature polarised localisation in cultured rat hippocampal neurons. I show that axonal surface polarity is initially established through axonally biased delivery of newly synthesized CB1R by the secretory pathway. Axonal surface polarity is subsequently enhanced and maintained by selective removal from the dendritic membrane and stable retention at the axonal surface.
This dual mechanism is mediated by the CB1R C-terminus and involves the Helix 9 (H9) domain. Removal of the H9 domain (CB1RΔH9) both increases secretory pathway delivery to dendrites and decreases surface stability. Furthermore, CB1RΔH9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1RWT.
A screen for H9 interacting proteins highlighted SH3-containing GRB2-like protein 3-interacting protein 1 (SGIP1), a protein linked to clathrin-mediated endocytosis. SGIP1 stabilises CB1R at the surface, likely through interaction with the H9 domain.
Together, these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process. This is important because defining the trafficking pathways and protein interactions that mediate axonal CB1R localisation could provide novel targets to enhance or reduce ECS signalling without the need to directly activate or block the receptor.
|Date of Award||23 Jan 2020|
|Supervisor||Jeremy M Henley (Supervisor)|