Single-incision laparoscopic surgery for benign multicystic mesothelioma of the peritoneum in a young man: A case report.

Benign multicystic peritoneal mesothelioma (BMPM) is a rare condition, particularly in men, and the preoperative diagnosis poses a challenge. Here, we present a case involving single-incision laparoscopic surgery (SILS) for BMPM in a 24-year-old man with a pelvic mass and a history of ulcerative colitis. Pelvic imaging revealed multifocal cysts, prompting the performance of SILS. The tumor was successfully resected with no residual lesions, and pathology confirmed the diagnosis of BMPM. This case represents the first documented instance of SILS being employed for BMPM in a man. BMPM, characterized by pelvic multifocal cysts, is a differential diagnosis, and SILS emerges as a viable option for both diagnosis and treatment.

Genome-wide analysis of DNA methylation in pseudomyxoma peritonei originated from appendiceal neoplasms.

INTRODUCTION: Pseudomyxoma peritonei (PMP) is a disease characterized by progressive accumulation of intraperitoneal mucinous ascites produced by neoplasms in the abdominal cavity. Since the prognosis of patients with PMP remain unsatisfactory, the development of effective therapeutic drug(s) is a matter of pressing concern. Genetic analyses of PMP have clarified the frequent activation of GNAS and/or KRAS. However, the involvement of global epigenetic alterations in PMPs has not been reported. METHODS: To clarify the genetic background of the 15 PMP tumors, we performed genetic analysis using AmpliSeq Cancer HotSpot Panel v2. We further investigated global DNA methylation in the 15 tumors and eight non-cancerous colonic epithelial cells using Methylation EPIC array BeadChip (Infinium 850k) containing a total of 865,918 probes. RESULTS: This is the first report of comprehensive DNA methylation profiles of PMPs in the world. We clarified that the 15 PMPs could be classified into at least two epigenotypes, unique methylation epigenotype (UME) and normal-like methylation epigenotype (NLME), and that genes associated with neuronal development and synaptic signaling may be involved in the development of PMPs. In addition, we identified a set of hypermethylation marker genes such as HOXD1 and TSPYL5 in the 15 PMPs. CONCLUSIONS: These findings may help the understanding of the molecular mechanism(s) of PMP and contribute to the development of therapeutic strategies for this life-threatening disease.

Clinical Effectiveness of Preoperative 18F-FDG PET/CT in Predicting Pathological Tumor Grade in Patients with Pseudomyxoma Peritonei Originating from Appendix: A Retrospective Cohort Study.

BACKGROUND: Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy is the standard treatment for patients with pseudomyxoma peritonei (PMP). In some malignancies, the standard uptake value of positron emission tomography with 2-deoxy-2-18F-fluoro-D-glucose integrated with computed tomography (18F-FDG PET/CT) is now accepted as a reliable indicator of neoplastic behavior. This study aimed to evaluate the association between the maximum standardized uptake value (SUVmax) and pathological grade in patients with PMP and to investigate the significance of SUVmax in the preoperative assessment of these patients. PATIENTS AND METHODS: In this retrospective single-center study, consecutively enrolled patients diagnosed with PMP of appendiceal origin underwent preoperative 18F-FDG PET/CT. SUVmax was calculated as the highest SUVmax value in the abdomen excluding the primary site. SUVmax was compared with the pathological grade (low or high grade) of PMP tumors according to the World Health Organization classification and further analyzed with respect to the estimated cutoff point, sensitivity, specificity, and receiver operating characteristic. RESULTS: In total, 160 patients were included. CRS was successfully performed in 93 patients and palliative debulking surgery in 67 patients. The pathological grade was high in 45 patients and low in 115. High-grade patients had a higher median SUVmax on 18F-FDG PET/CT than did low-grade patients (3.83 versus 2.34, p < 0.001). The highest area under the curve was 0.81, with a sensitivity of 77.8%, specificity of 72.3%, and cutoff point of 2.63. CONCLUSION: This study suggests that the SUVmax of preoperative 18F-FDG PET/CT is associated with the pathological grade in patients with PMP.

A case of pseudomyxoma peritonei arising from a perforated intraductal papillary mucinous neoplasm that underwent cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

Pseudomyxoma peritonei (PMP) of pancreatic origin arising from an intraductal papillary mucinous neoplasm (IPMN) is rare. Cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) has been established as the optimal treatment for PMP. However, the benefits and safety of CRS with HIPEC for treating PMP of pancreatic origin remain unclear. Herein, we describe a case of PMP of pancreatic origin that was treated with CRS and HIPEC without postoperative complications. A 75-year-old woman was referred to our department. Computed tomography (CT) revealed a multilocular cystic tumor in the pancreatic tail, notable mucinous ascites in the abdominal cavity, and scalloping of the liver and spleen. CT did not reveal the appendix, and the ovaries were normal in size. The patient was diagnosed with PMP of pancreatic origin, and CRS and HIPEC were performed. Intraoperatively, the pancreatic tumor was perforated, and there was a large amount of mucinous ascites. We performed distal pancreatectomy in addition to CRS and HIPEC, with no intraoperative complications. The postoperative course was uneventful, and the patient survived after 6 months without recurrence. CRS with HIPEC may be a feasible treatment option for PMP of pancreatic origin.

Subtle Structural Modification of a Synthetic Cannabinoid Receptor Agonist Drastically Increases its Efficacy at the CB1 Receptor.

The emergence of synthetic cannabinoid receptor agonists (SCRAs) as illicit psychoactive substances has posed considerable public health risks, including fatalities. Many SCRAs exhibit much higher efficacy and potency compared with the phytocannabinoid Δ9-tetrahydrocannabinol (THC) at the cannabinoid receptor 1 (CB1R), leading to dramatic differences in signaling levels that can be toxic. In this study, we investigated the structure-activity relationships of aminoalkylindole SCRAs at CB1Rs, focusing on 5F-pentylindoles containing an amide linker attached to different head moieties. Using in vitro bioluminescence resonance energy transfer assays, we identified a few SCRAs exhibiting significantly higher efficacy in engaging the Gi protein and recruiting ß-arrestin than the reference CB1R full agonist CP55940. Importantly, the extra methyl group on the head moiety of 5F-MDMB-PICA, as compared to that of 5F-MMB-PICA, led to a large increase in efficacy and potency at the CB1R. This pharmacological observation was supported by the functional effects of these SCRAs on glutamate field potentials recorded in hippocampal slices. Molecular modeling and simulations of the CB1R models bound with both of the SCRAs revealed critical structural determinants contributing to the higher efficacy of 5F-MDMB-PICA and how these subtle differences propagated to the receptor-G protein interface. Thus, we find that apparently minor structural changes in the head moiety of SCRAs can cause major changes in efficacy. Our results highlight the need for close monitoring of the structural modifications of newly emerging SCRAs and their potential for toxic drug responses in humans.

A subtle structural modification of a synthetic cannabinoid receptor agonist drastically increases its efficacy at the CB1 receptor.

The emergence of synthetic cannabinoid receptor agonists (SCRAs) as illicit psychoactive substances has posed considerable public health risks that include fatalities. Many SCRAs exhibit much higher efficacy and potency, compared with the phytocannabinoid Δ9-tetrahydrocannabinol (THC), at the cannabinoid receptor 1 (CB1R), a G protein-coupled receptor involved in modulating neurotransmitter release. In this study, we investigated structure activity relationships (SAR) of aminoalkylindole SCRAs at CB1Rs, focusing on 5F-pentylindoles containing an amide linker attached to different head moieties. Using in vitro bioluminescence resonance energy transfer (BRET) assays, we identified a few of SCRAs exhibiting significantly higher efficacy in engaging the Gi protein and recruiting ß-arrestin than the reference CB1R full agonist CP55940. Importantly, adding a methyl group at the head moiety of 5F-MMB-PICA yielded 5F-MDMB-PICA, an agonist exhibiting a large increase in efficacy and potency at the CB1R. This pharmacological observation was supported by a functional assay of the effects of these SCRAs on glutamate field potentials recorded in hippocampal slices. Molecular modeling and simulations of the CB1R bound with either of the SCRAs revealed critical structural determinants contributing to the higher efficacy of 5F-MDMB-PICA, and how these subtle differences propagated to the receptor-G protein interface. Thus, we find that apparently minor structural changes in the head moiety of SCRAs can cause major changes in efficacy. Our results highlight the need for close monitoring of structural modifications of newly emerging SCRAs and their potential for toxic drug responses in humans.

Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G protein subunits.

G-protein-coupled receptors (GPCRs) mediate neuromodulation through the activation of heterotrimeric G proteins (Gαßγ). Classical models depict that G protein activation leads to a one-to-one formation of Gα-GTP and Gßγ species. Each of these species propagates signaling by independently acting on effectors, but the mechanisms by which response fidelity is ensured by coordinating Gα and Gßγ responses remain unknown. Here, we reveal a paradigm of G protein regulation whereby the neuronal protein GINIP (Gα inhibitory interacting protein) biases inhibitory GPCR responses to favor Gßγ over Gα signaling. Tight binding of GINIP to Gαi-GTP precludes its association with effectors (adenylyl cyclase) and, simultaneously, with regulator-of-G-protein-signaling (RGS) proteins that accelerate deactivation. As a consequence, Gαi-GTP signaling is dampened, whereas Gßγ signaling is enhanced. We show that this mechanism is essential to prevent the imbalances of neurotransmission that underlie increased seizure susceptibility in mice. Our findings reveal an additional layer of regulation within a quintessential mechanism of signal transduction that sets the tone of neurotransmission.

Highly Potent and Selective Dopamine D4 Receptor Antagonists Potentially Useful for the Treatment of Glioblastoma.

To better understand the role of dopamine D4 receptor (D4R) in glioblastoma (GBM), in the present paper, new ligands endowed with high affinity and selectivity for D4R were discovered starting from the brain penetrant and D4R selective lead compound 1-(3-(4-phenylpiperazin-1-yl)propyl)-3,4-dihydroquinolin-2(1H)-one (6). In particular, the D4R antagonist 24, showing the highest affinity and selectivity over D2R and D3R within the series (D2/D4 = 8318, D3/D4 = 3715), and the biased ligand 29, partially activating D4R Gi-/Go-protein and blocking ß-arrestin recruitment, emerged as the most interesting compounds. These compounds, evaluated for their GBM antitumor activity, induced a decreased viability of GBM cell lines and primary GBM stem cells (GSC#83), with the maximal efficacy being reached at a concentration of 10 µM. Interestingly, the treatment with both compounds 24 and 29 induced an increased effect in reducing the cell viability with respect to temozolomide, which is the first-choice chemotherapeutic drug in GBM.

Association of intraoperative gross hematuria with acute kidney injury after cytoreductive surgery.

Objectives: Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) has been established in the management of peritoneal carcinomatosis. Although it is still necessary to take adequate measures against major postoperative complications including acute kidney injury (AKI), consensus is lacking on how to assess and stratify risk for patients with postoperative AKI after CRS-HIPEC. The aim of this retrospective cohort study was to investigate the association of intraoperative gross hematuria as a surrogate marker of ureter injury with postoperative AKI incidence. Methods: This retrospective cohort study investigated patients without impaired preoperative kidney function who underwent CRS-HIPEC at a single referral center, and evaluated the relationship between intraoperative gross hematuria and incidence of postoperative AKI as defined by the Kidney Disease Improving Global Outcomes practice guidelines. Logistic regression analysis was performed to calculate the odds ratio of intraoperative gross hematuria for AKI, adjusting for confounding factors and other risk factors for AKI. Results: We enrolled 185 patients (males, 37%). Twenty-five patients developed intraoperative gross hematuria. Postoperative AKI occurred in 10 (40%) of 25 patients with hematuria and 28 (17.5%) of 160 patients without hematuria. The crude odds ratio for exposure to hematuria was 3.14 (95% confidence interval, 1.30-7.60; p=0.020) for postoperative AKI. Adjusted odds ratio as estimated by multivariate logistic regression was 4.57 (95% confidence interval, 1.55-13.45; p=0.006). Conclusions: Intraoperative gross hematuria is significantly associated with postoperative AKI incidence after CRS-HIPEC.

Prognostic value of 18F-FDG PET/CT with texture analysis in patients with rectal cancer treated by surgery.

PURPOSE: The aim of this study was to evaluate the ability of texture analysis using pretreatment 18F-FDG PET/CT to predict prognosis in patients with surgically treated rectal cancer. METHODS: We analyzed 94 patients with pathologically proven rectal cancer who underwent pretreatment 18F-FDG PET/CT and were subsequently treated with surgery. The volume of interest of the primary tumor was defined using a threshold of 40% of the maximum standardized uptake value (SUVmax), and conventional (SUVmax, metabolic tumor volume [MTV], total lesion glycolysis [TLG]) and textural PET features were extracted. Harmonization of PET features was performed with the ComBat method. The study endpoints were overall survival (OS) and progression-free survival (PFS), and the prognostic value of PET features was evaluated by Cox regression analysis. RESULTS: In the follow-up period (median 41.7 [interquartile range, 30.5-60.4] months), 21 (22.3%) and 30 (31.9%) patients had cancer-related death or disease progression, respectively. Univariate analysis revealed a significant association of (1) MTV, TLG, and gray-level co-occurrence matrix (GLCM) entropy with OS; and (2) SUVmax, MTV, TLG, and GLCM entropy with PFS. In multivariate analysis including clinical characteristics, GLCM entropy (≥ 2.13) was the only relevant prognostic PET feature for poor OS (hazard ratio [HR]: 4.16, p = 0.035) and PFS (HR: 2.70, p = 0.046). CONCLUSION: GLCM entropy, which indicates metabolic intratumoral heterogeneity, was an independent prognostic factor in patients with surgically treated rectal cancer. Compared with conventional PET features, GLCM entropy has better predictive value and shows potential to facilitate precision medicine.

A target-agnostic screen identifies approved drugs to stabilize the endoplasmic reticulum-resident proteome.

Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.

Chirality of Novel Bitopic Agonists Determines Unique Pharmacology at the Dopamine D3 Receptor.

The dopamine D2/D3 receptor (D2R/D3R) agonists are used as therapeutics for Parkinson's disease (PD) and other motor disorders. Selective targeting of D3R over D2R is attractive because of D3R's restricted tissue distribution with potentially fewer side-effects and its putative neuroprotective effect. However, the high sequence homology between the D2R and D3R poses a challenge in the development of D3R selective agonists. To address the ligand selectivity, bitopic ligands were designed and synthesized previously based on a potent D3R-preferential agonist PF592,379 as the primary pharmacophore (PP). This PP was attached to various secondary pharmacophores (SPs) using chemically different linkers. Here, we characterize some of these novel bitopic ligands at both D3R and D2R using BRET-based functional assays. The bitopic ligands showed varying differences in potencies and efficacies. In addition, the chirality of the PP was key to conferring improved D3R potency, selectivity, and G protein signaling bias. In particular, compound AB04-88 exhibited significant D3R over D2R selectivity, and G protein bias at D3R. This bias was consistently observed at various time-points ranging from 8 to 46 min. Together, the structure-activity relationships derived from these functional studies reveal unique pharmacology at D3R and support further evaluation of functionally biased D3R agonists for their therapeutic potential.

A case of pseudomyxoma peritonei successfully treated with trifluridine/tipiracil (TAS-102) and bevacizumab after palliative debulking surgery.

Pseudomyxoma peritonei is a rare disease with a reported prevalence of about 1-3 per million people annually. Cytoreductive surgery and perioperative hyperthermic intraperitoneal chemotherapy are considered as treatment options improving disease control or long-term survival. However, for patients with incomplete cytoreduction or debulking surgery, outcomes are significantly poorer compared with patients who have obtained complete or optimal cytoreduction. In cases of high-grade pseudomyxoma peritonei that are considered inoperable and/or unresectable, combination chemotherapy regimen with a neo-angiogenesis inhibitor such as bevacizumab is recommended. In this report, a 62-year-old Japanese man presented with abdominal distention. Examination of ascites demonstrated a jelly-like consistency and peritoneal pseudomyxoma was suspected. To relieve progressive symptoms, palliative debulking surgery with total colectomy was performed. Postoperative pathology confirmed high-grade appendiceal mucinous neoplasm with high-grade pseudomyxoma peritonei. In our case, combination chemotherapy with trifluridine/tipiracil (TAS-102) and bevacizumab was initiated after palliative debulking surgery. As a result, carcinoembryonic antigen level was kept stable and the volume of ascites remained almost the same as at the beginning of treatment for more than 2 years. In conclusion, combination chemotherapy comprising TAS-102 and bevacizumab in patients with palliative debulking could be a useful option for patients with high-grade mucinous appendiceal neoplasm and high-grade pseudomyxoma peritonei.

The National Health Service (NHS) response to the COVID-19 pandemic: a colorectal surgeons experience in the UK.

The UK government was arguably slow to take action against the COVID-19 pandemic. However, since switching their policy from "mitigation" to "suppression", swift changes have been implemented to all aspects of life. In this unprecedented crisis healthcare has been on the battlefront across the globe. Every effort has been made in the UK to stop the National Health Service (NHS) from being overwhelmed, leading to the national slogan: "Stay at home. Protect the NHS. Save lives". In this article, a consultant general and colorectal surgeon in Southampton reports on the NHS response to the COVID-19 pandemic.

Cytoreductive Surgery Combined with Hyperthermic Intraperitoneal Chemotherapy for Peritoneal Metastasis from Colorectal Cancer.

Peritoneal metastasis from colorectal cancer (PM-CRC) is used to be considered a systemic and fatal condition; however, it has been growingly accepted that PM-CRC can still be local disease rather than systemic disease as analogous to liver or lung metastasis. Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is now considered an optimal treatment for PM-CRC with accumulating evidence. There is a good reason that CRS + HIPEC, widely accepted as a standard of care for pseudomyxoma peritonei (PMP), could be a viable option for PM-CRC given a similarity between PM-CRC and PMP. Recent years have also seen that modern systemic chemotherapy with or without molecular targeted agents can be effective for PM-CRC. It is possible that neoadjuvant or adjuvant chemotherapy combined with CRS + HIPEC could further improve outcomes. Patient selection, utilizing modern images and increasingly laparoscopy, is crucial. Particularly, diagnostic laparoscopy is likely to play a significant role in predicting the likelihood of achieving complete cytoreduction and assessing the peritoneal cancer index score.

Immunohistochemical staining as supportive diagnostic tool for pseudomyxoma peritonei arising from intraductal papillary mucinous neoplasm: A report of two cases and literature review.

BACKGROUND/OBJECTIVES: Pseudomyxoma peritonei (PMP) arising from an intraductal papillary mucinous neoplasm of the pancreas (IPMN) is a rare condition. The diagnosis of IPMN as the origin of PMP is mainly inferred from the clinical course and the exclusion of PMP from other organs. The pathological diagnosis has not yet been established. To evaluate the usefulness of immunohistochemical staining for the diagnosis of the primary lesion of PMP as IPMN. METHODS: There are 2 cases of PMP arising from IPMN between March 2010 and December 2019 at National Center for Global Health and Medicine. A PubMed search that reported PMP arising from IPMN identified 16 additional cases. Diagnostic methods and clinicopathological features of 18 cases were compared. RESULTS: Four cases including our two cases used immunohistochemical staining for the diagnosis of PMP arising from IPMN. The correspondence of the immunohistochemical staining between PMP and IPMN was shown in the three cases including previously reported two cases and one of our two cases to identify the primary lesion of PMP as IPMN. In addition, we revealed that the comparison of the immunostaining pattern of PMP with the representative immunostaining pattern of the candidate primary lesions is helpful for the diagnosis of the primary lesion of PMP. CONCLUSIONS: Immunohistochemical staining is helpful to identify the primary lesion of PMP as IPMN.

Positive Allosteric Modulation of the 5-HT1A Receptor by Indole-Based Synthetic Cannabinoids Abused by Humans.

The nonmedical (i.e., recreational) misuse of synthetic cannabinoids (SCs) is a worldwide public health problem. When compared to cannabis, the misuse of SCs is associated with a higher incidence of serious adverse effects, suggesting the possible involvement of noncannabinoid sites of action. Here, we find that, unlike the phytocannabinoid Δ9-tetrahydrocannabinol, the indole-moiety containing SCs, AM2201 and JWH-018, act as positive allosteric modulators (PAMs) at the 5-HT1A receptor (5-HT1AR). This suggests that some biological effects of SCs might involve allosteric interactions with 5-HT1ARs. To test this hypothesis, we examined effects of AM2201 on 5-HT1AR agonist-activated G protein-coupled inwardly rectifying potassium channel currents in neurons in vitro and on the hypothermic response to 5-HT1AR stimulation in mice lacking the cannabinoid receptor 1. We found that both 5-HT1AR effects were potentiated by AM2201, suggesting that PAM activity at 5-HT1AR may represent a novel noncannabinoid receptor mechanism underlying the complex profile of effects for certain SCs.

Pseudomyxoma peritonei: visceral scalloping on CT is a predictor of recurrence after complete cytoreductive surgery.

OBJECTIVES: Pseudomyxoma peritonei (PMP) is characterized by peritoneal dissemination of gelatinous ascites following rupture of a mucinous tumor. Treatment by cytoreductive surgery (CRS) has improved its prognosis. Although visceral scalloping, notably liver scalloping, on computed tomography (CT) is a typical feature of PMP, its prognostic value remains unknown. We aimed to investigate the efficacy of liver scalloping in predicting recurrence in PMP patients. METHODS: Among 159 consecutive patients with PMP who had contrast-enhanced CT between September 2012 and December 2018, 64 treatment-naïve patients who subsequently underwent CRS with complete resection (i.e., completeness of cytoreduction score (CC)-0 or CC-1), were included in analysis. Presence of liver scalloping and maximum thickness of mucin deposition at the liver surface were evaluated on CT. Disease-free survival (DFS) was determined based on the combination of postoperative CT features and tumor marker values. RESULTS: Median follow-up was 24.3 months. CT revealed liver scalloping in 40/64 (63.4%) patients. Kaplan-Meier analysis showed significantly shorter DFS in patients with scalloping than in those without (p = 0.001; hazard ratio, 4.3). In patients with scalloping, greater mucin deposition (thickness ≥ 20 mm) significantly correlated with poorer DFS (p = 0.042). In multivariate Cox proportional hazards regression including CC status, pathologic type, and tumor markers, the presence of scalloping independently and significantly correlated with DFS (p = 0.031). CONCLUSIONS: Liver scalloping was an independent predictor even after adjusting for clinical covariates. The presence of liver scalloping can lead to a high recurrence rate after CRS. KEY POINTS: • The presence of liver scalloping is a prognostic factor independent of histological grade and tumor markers. • Greater mucin deposition (thickness ≥ 20 mm at the liver surface) is associated with higher recurrence rates in patients with liver scalloping.

The Glu102 mutation disrupts higher-order oligomerization of the sigma 1 receptor.

The sigma 1 receptor (σ1R) is a unique endoplasmic reticulum membrane protein. Its ligands have been shown to possess therapeutic potential for neurological and substance use disorders among others. The E102Q mutation of σ1R has been found to elicit familial cases of amyotrophic lateral sclerosis (ALS). Despite reports of its downstream signaling consequences, the mechanistic details of the functional impact of E102Q at molecular level are not clear. Here, we investigate the molecular mechanism of the E102Q mutation with a spectrum of biochemical, biophysical, and pharmacological approaches. Our analysis of the interaction network of σ1R indicates that a set of residues near E102 is critical for the integrity of C-terminal ligand-binding domain. However, this integrity is not affected by the E102Q and E102A mutations, which is confirmed by the radioligand binding results. Instead, the E102 mutations disrupt the connection between the C-terminal domain and the N-terminal transmembrane helix (NT-helix). Results from bioluminescence resonance energy transfer and western blot assays demonstrate that these mutations destabilize higher-order σ1R oligomers, while our molecular dynamics simulations based on a σ1R crystal structure reveal a potential mechanism by which the mutations perturb the NT-helix dynamics. Thus, we propose that E102 is at a critical position in propagating the effects of ligand binding from the C-terminal domain to the NT-helix, while the latter may be involved in forming alternative oligomer interfaces, separate from the previously reported trimer interface. Together, these results provide the first account of the molecular mechanism of σ1R dysfunction caused by E102Q.

A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity.

Signaling bias is the propensity for some agonists to preferentially stimulate G protein-coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein-biased agonist of the D2 dopamine receptor (D2R) that results in impaired ß-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with ß-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired ß-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the ß2-adrenergic receptor (ß2R) to build ß2R-WT and ß2R-Y1995.38A models in complex with the full ß2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in ß2R-Y1995.38A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in ß2R-Y1995.38A, which is predicted to affect its interactions with ß-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.