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 β-arrestin. It has been postulated that phosphorylation of two clusters of serine/threonine residues, ⁴²⁵SMGDS⁴²⁹ and ⁴⁶⁰TMSVSTDTS⁴⁶⁸, 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 Gβγ 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 Gβγ 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.

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.

Alternative splicing can often result in the expression of distinct protein isoforms from a single gene, with specific composition and properties. SH3-containing GRB2-like protein 3-interacting protein 1 (Sgip1) is a brain-enriched protein that regulates clathrin-mediated endocytosis and interferes with the internalization of cannabinoid receptor 1. Several research groups have studied the physiological importance of Sgip1, and four Sgip1 protein isoforms have been described to date, while the NCBI Gene database predicts the expression of 20 splice variants from the Sgip1 gene in mice. In this work, we cloned 15 Sgip1 splice variants from the mouse brain, including 11 novel splice variants. The cloned splice variants differed in exon composition within two Sgip1 regions: the membrane phospholipid-binding domain and the proline-rich region. All the Sgip1 splice isoforms had similar stability and comparable ability to inhibit the internalization of cannabinoid receptor 1. None of the isoforms influenced the internalization of the µ-opioid receptor. We confirm the expression of Sgip1 splice variants described in previous studies or predicted in silico. Our data provide a basis for further studies exploring the significance of Sgip1 splicing, and we suggest a new classification of Sgip1 splice variants to unify their nomenclature.

The evolutionarily conserved Swi5-Sfr1 complex plays an important role in homologous recombination, a process crucial for the maintenance of genomic integrity. Here, we purified Schizosaccharomyces pombe Swi5-Sfr1 complex from meiotic cells and analyzed it by mass spectrometry. Our analysis revealed new phosphorylation sites on Swi5 and Sfr1. We found that mutations that prevent phosphorylation of Swi5 and Sfr1 do not impair their function but swi5 and sfr1 mutants encoding phosphomimetic aspartate at the identified phosphorylation sites are only partially functional. We conclude that during meiosis, Swi5 associates with Sfr1 and both Swi5 and Sfr1 proteins are phosphorylated. However, the functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined.

Parkinson’s disease (PD) is associated with systemic proteostatic failure, chronic neuroinflammation, and mitochondrial abnormalities linked to severe oxidative stress. This exploratory longitudinal study mapped the deep-coverage plasma proteome (~6000 proteins via SOMAmer technology) in 32 patients with mild-to-moderate PD following a 21-day intensive, multimodal physiological stimulus framework. This hormetic intervention combined balneo- and hydropinic therapy (incorporating mineral water with 7.48 mg/L H₂S and peloid mud with 6.14% S²⁻ of dry matter), neurophysiotherapy, a neuroprotective MIND diet, mindfulness meditation, and clinical aromatherapy. Systems-level pathway analysis was leveraged to evaluate alterations in functional biological networks. Gene Set Enrichment Analysis (GSEA) resolved a negative enrichment of the Parkinson’s disease pathway hsa05012 (FDR = 0.0053). Network topology modeling revealed a highly coordinated proteostatic and antioxidant alteration pattern involving interferon-gamma (IFN-γ) and polyubiquitin-B (UBB) protein clusters (p = 2.2 × 10⁻⁵), accompanied by the downregulation of protein clearance markers (UBB, p62, and UCHL1). Concurrently, upregulation of the phase-II detoxification modifier sulfotransferase 1C2 (SULT1C2; log₂FC = 0.42) and a reduction of circulating mitochondrial respiratory subunits (NDUFB6, COX5A, NDUFB11) were observed. These exploratory findings may suggest that multimodal hormetic interventions trigger systemic, redox-responsive changes in protein signatures. However, because the distinct volatile chemical and organoleptic properties of the intervention prevent a sham control, these open-label findings must be treated as hypothesis-generating. These tentative models require verification in future parallel-controlled trials before definitive causal interpretations can be established.