SGIP1 Deletion in Mice Attenuates Mechanical Hypersensitivity Elicited by Inflammation.

Background: Activation of cannabinoid receptor 1 (CB1R) in the nervous system modulates the processing of acute and chronic pain. CB1R activity is regulated by desensitization and internalization. SH3-containing GRB2-like protein 3-interacting protein 1 (SGIP1) inhibits the internalization of CB1R. This causes increased and prolonged association of the desensitized receptor with G protein-coupled receptor kinase 3 (GRK3) and beta-arrestin on the cell membrane and results in decreased activation of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Genetic deletion of SGIP1 in mice leads to altered CB1R-related functions, such as decreased anxiety-like behaviors, modified cannabinoid tetrad behaviors, reduced acute nociception, and increased sensitivity to analgesics. In this work, we asked if deletion of SGIP1 affects chronic nociception and analgesic effect of Δ9-tetrahydrocannabinol (THC) and WIN 55,212-2 (WIN) in mice. Methods: We measured tactile responses of hind paws to increasing pressure in wild-type and SGIP1 knock-out mice. Inflammation in the paw was induced by local injection of carrageenan. To determine the mechanical sensitivity, the paw withdrawal threshold (PWT) was measured using an electronic von Frey instrument with the progression of the applied force. Results: The responses to mechanical stimuli varied depending on the sex, genotype, and treatment. SGIP1 knock-out male mice exhibited lower PWT than wild-type males. On the contrary, the female mice exhibited comparable PWT. Following THC or WIN treatment in male mice, SGIP1 knock-out males exhibited PWT lower than wild-type males. THC treatment in SGIP1 knock-out females resulted in PWT higher than after THC treatment of wild-type females. However, SGIP1 knock-out and wild-type female mice exhibited similar PWT after WIN treatment. Conclusions: We provide evidence that SGIP1, possibly by interacting with CB1R, is involved in processing the responses to chronic pain. The absence of SGIP1 results in enhanced sensitivity to mechanical stimuli in males, but not females. The antinociceptive effect of THC is superior to that of WIN in SGIP1 knock-out mice in the carrageenan-induced model of chronic pain.

Hexahydrocannabinol (HHC) and Δ9-tetrahydrocannabinol (Δ9-THC) driven activation of cannabinoid receptor 1 results in biased intracellular signaling.

The Cannabis sativa plant has been used for centuries as a recreational drug and more recently in the treatment of patients with neurological or psychiatric disorders. In many instances, treatment goals include relief from posttraumatic disorders, anxiety, or to support treatment of chronic pain. Ligands acting on cannabinoid receptor 1 (CB1R) are also potential targets for the treatment of other health conditions. Using an evidence-based approach, pharmacological investigation of CB1R agonists is timely, with the aim to provide chronically ill patients relief using well-defined and characterized compounds from cannabis. Hexahydrocannabinol (HHC), currently available over the counter in many countries to adults and even children, is of great interests to policy makers, legal administrators, and healthcare regulators, as well as pharmacologists. Herein, we studied the pharmacodynamics of HHC epimers, which activate CB1R. We compared their key CB1R-mediated signaling pathway activities and compared them to the pathways activated by Δ9-tetrahydrocannabinol (Δ9-THC). We provide evidence that activation of CB1R by HHC ligands is only broadly comparable to those mediated by Δ9-THC, and that both HHC epimers have unique properties. Together with the greater chemical stability of HHC compared to Δ9-THC, these molecules have a potential to become a part of modern medicine.

SGIP1 in axons prevents internalization of desensitized CB1R and modifies its function.

In the central nervous system (CNS), cannabinoid receptor 1 (CB1R) is preferentially expressed in axons where it has a unique property, namely resistance to agonist-driven endocytosis. This review aims to summarize what we know about molecular mechanisms of CB1R cell surface stability in axonal compartments, how these impact CB1R signaling, and to consider their physiological consequences. This review then focuses on a potential candidate for maintaining axonal CB1R at the cell surface, Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1 (SGIP1). SGIP1 may contribute to the polarized distribution of CB1R and modify its signaling in axons. In addition, deletion of SGIP1 results in discrete behavioral changes in modalities controlled by the endocannabinoid system in vivo. Several drugs acting directly via CB1R have important therapeutic potential, however their adverse effects limit their clinical use. Future studies might reveal chemical approaches to target the SGIP1-CB1R interaction, with the aim to exploit the endocannabinoid system pharmaceutically in a discrete way, with minimized undesired consequences.

SGIP1 modulates kinetics and interactions of the cannabinoid receptor 1 and G protein-coupled receptor kinase 3 signalosome.

Cannabinoid receptor 1 (CB1R), a G protein-coupled receptor, plays a fundamental role in synaptic plasticity. Abnormal activity and deregulation of CB1R signaling result in a broad spectrum of pathological conditions. CB1R signaling is regulated by receptor desensitization including phosphorylation of residues within the intracellular C terminus by G protein-coupled receptor kinases (GRKs) that may lead to endocytosis. Furthermore, CB1R signaling is regulated by the protein Src homology 3-domain growth factor receptor-bound 2-like (SGIP1) that hinders receptor internalization, while enhancing CB1R association with ß-arrestin. It has been postulated that phosphorylation of two clusters of serine/threonine residues, 425 SMGDS429 and 460 TMSVSTDTS468 , within the CB1R C-tail controls dynamics of the association between receptor and its interaction partners involved in desensitization. Several molecular determinants of these events are still not well understood. We hypothesized that the dynamics of these interactions are modulated by SGIP1. Using a panel of CB1Rs mutated in the aforementioned serine and threonine residues, together with an array of Bioluminescence energy transfer-based (BRET) sensors, we discovered that GRK3 forms complexes with Gßγ subunits of G proteins that largely independent of GRK3's interaction with CB1R. Furthermore, CB1R interacts only with activated GRK3. Interestingly, phosphorylation of two specific residues on CB1R triggers GRK3 dissociation from the desensitized receptor. SGIP1 increases the association of GRK3 with Gßγ subunits of G proteins, and with CB1R. Altogether, our data suggest that the CB1R signalosome complex is dynamically controlled by sequential phosphorylation of the receptor C-tail and is also modified by SGIP1.