Rithwik Ramachandran

Kindlins are essential for integrin activation and mechanoresponses. In the epidermis, inherited loss-of-function of the FERMT1 gene, which encodes Kindlin-1, increases the risk of developing cutaneous squamous cell carcinoma (cSCC). Notably, the role of Kindlin-1 in epidermal tumours has remained virtually unexplored. In this study, we analyzed the consequences of FERMT1 inactivation in a cSCC cell model. Kindlin-1-depleted cells exhibited decreased number and size of focal adhesions, as well as alterations in actin stress fibres and the F-actin perinuclear cap. Significantly, FERMT1 inactivation also resulted in reduced nuclear volume and total surface area. These abnormalities were not ameliorated by exogenous expression of Kindlin-2 or a Kindlin-1 mutant incapable of binding to integrins. In contrast, nuclear dimensions in Kindlin-1-deficient cells were restored in the presence of a constitutively active RhoA protein. These findings are consistent with the concept that Kindlin-1 plays an essential role mediating integrin signalling and RhoA activation to modulate perinuclear actin filaments and nuclear morphology.

The Concise Guide to Pharmacology 2025/26 marks the seventh edition in this series of biennial publications in the British Journal of Pharmacology. Presented in landscape format, the guide provides a comparative overview of the pharmacology of drug target families. The concise nature of the Concise Guide refers to the style of presentation, being clear, accessible, and well‐structured, rather than the scope of the content, which spans approximately 500 pages. The Concise Guide summarises the key pharmacological properties of around 1900 human drug targets, and nearly 7000 interactions, involving around 4400 ligands. While the content is a substantially condensed version of the more detailed information and links available at the www.guidetopharmacology.org website, the printed guide serves as a permanent, citable, point‐in‐time record, that remains stable despite ongoing updates to the online database. The full contents of this publication can be found at https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70230 . The Concise Guides provide expert‐curated recommendations of ‘Gold Standard’ selective pharmacological tools, available either commercially or as donations, which enable the identification of individual drug targets or families of drug targets. While the Concise Guide offers a more streamlined overview, more comprehensive information, including detailed pharmacological profiles and links to multiple online databases, is available through the Guide to Pharmacology website. The 2025/26 edition of the Concise Guide is based on material current as of mid‐2025, and supersedes all previous editions, including the 2023/24 Guide, and earlier Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), and as such provides official IUPHAR classification and nomenclature for human drug targets, where applicable. G protein‐coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. Each section includes nomenclature guidance, concise summaries, information of the best available pharmacological tools, key references, and suggestions for further reading.

Calcium (Ca ²⁺ ) is critical for normal cell function, and several protein networks are required for Ca ²⁺ signaling. In the pancreas, regulated changes in cytosolic Ca ²⁺ allow for the exocytosis of zymogen granules, and altered Ca ²⁺ signaling underlies pancreatic pathologies. Previously, our laboratory showed a pancreas‐specific isoform of secretory pathway Ca ²⁺ ‐ATPase 2 (also known as calcium‐transporting ATPase type 2C member 2; SPCA2), termed SPCA2C (gene name Atp2c2c , C‐terminally truncated form), affects multiple pathways involved in Ca ²⁺ homeostasis. The goal of this study was to define the SPCA2C interactome that contributes to these processes. Using proximity‐dependent biotin identification, BioID, we expressed SPCA2C‐BirA* ^HA in HEK293 cells with constitutive calcium release‐activated calcium channel protein 1 ( Orai1 ) expression. A total of 150 high‐confidence interacting proteins for SPCA2C were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analyses supported the localization of SPCA2C to the endoplasmic reticulum, and that it functions in Ca ²⁺ signaling and vesicular transport. Stromal interaction molecule 1 (STIM1) and coiled‐coil domain‐containing protein 47 (CCDC47; also known as PAT complex subunit CCDC47) were among several proteins we confirmed as strong interactors through co‐immunoprecipitation (co‐IP). Co‐expression of Atp2c2c and CCDC47 in HEK‐Orai1 ^YFP cells increased store‐operated calcium entry (SOCE) and resting cytosolic Ca ²⁺ levels compared with the expression of either protein alone. We further teased out the domain determinants of CCDC47 interactions with SPCA2C, STIM1, and Orai1 using co‐IP and co‐localization experiments. CCDC47 localized with SPCA2C and the key store‐operated Ca ²⁺ entry mediators STIM1 and Orai1 but did not interact with the long SPCA2 isoform. These interactions were dependent on the presence of the CCDC47 coiled‐coil or accessible transmembrane domains. Overall, we define several previously unknown protein interactions for SPCA2C, and suggest that CCDC47 may be involved in the coiled‐coil interplay that underlies STIM1 and Orai1‐mediated Ca ²⁺ entry.

The elevation of glucocorticoids is a hallmark of stress, arising from the integration of rapid neuronal signals into sustained hormonal outputs. A key interface for this neuroendocrine signal translation is corticotropin releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN), which release CRH at the median eminence (ME). We recently discovered that CRH _PVN neurons exhibit a characteristic shift in firing patterns from rhythmic short-bursts (low-activity state) to tonic firing (high-activity state) in response to stress. This raised a critical question: how are these distinct firing patterns are integrated into slower, sustained hormonal outputs at neuroendocrine terminals? Here, we implemented optical approaches to detect CRH release ex vivo and in vivo. Using newly developed sniffer cells for CRH, we measured CRH release at ME ex vivo , triggered by the distinct, in vivo -like firing patterns. Our results demonstrated that the primary determinant of neuroendocrine CRH release was the firing rate sustained over the timescale of seconds, with little contribution from specific firing patterns. These results collaborated with second-scale increase in firing rate triggered by stress stimuli. Additionally, we recorded the dynamics of CRH at the ME in the freely moving mice using genetically-encoded GPCR-activation based (GRAB) sensors for CRH. Foot shock stress triggered transient, time-locked increases in CRH release on the timescale of seconds. Importantly, these second-scale CRH pulses, when elicited during repeated foot shocks, were integrated over minutes to scale downstream hormone releases. Together, our data revealed critical roles of second-scale dynamics in CRH _PVN neuron activity for the neuroendocrine translation of stress signals.

The 8th-helix (H8) and carboxyl terminal tail (CT) of GPCRs are crucial for interactions with intracellular signaling molecules and regulatory proteins. We investigated how H8 and CT residues influence signaling in PAR4, a tethered-ligand activated GPCR. Our analysis revealed that PAR4 activation by thrombin or AYPGKF-NH2 stimulates β-arrestin-1/-2 recruitment and activates multiple Gα proteins (Gαq, Gα11, Gαz, Gα15, Gα12, Gα13, Gαi1-3, GαoA/B). Mutation of the H8 sequence (R ³⁵² AGLFQRS ³⁵⁹ ) significantly reduced G protein activation and β-arrestin recruitment, with residues Leu ³⁵⁵ -Glu ³⁵⁷ being particularly important. Mutations of specific lysine residues in H8 and CT also impaired signaling. Additionally, we identified a crucial TM7-H8 interaction and found that CT phosphorylation sites regulate β-arrestin recruitment. Finally, using AlphaFold3 we predicted interactions between PAR4, β-arrestins, and G proteins, revealing novel receptor regulatory sites in the intracellular loops, transmembrane domains, H8 and CT.

Adhesion GPCRs (aGPCRs), a family of 33 receptors, regulate crucial physiological processes through a unique self-activation mechanism. We employed AlphaFold, a state-of-the-art AI system, to predict 3D structures of aGPCRs in active and inactive conformations. Full-length models were obtained from the AlphaFold Protein Structure Database, while tethered-ligand (TL) exposed structures were predicted using AlphaFold. Comparison with available experimentally solved structures were made to validate accuracy of Alphafold aGPCR model. For receptors without experimentally solved structures, we compare structures of inactive (TL masked) and active (TL exposed) aGPCRs. In the active structures, the tethered-ligand was docked in the transmembrane bundle for several receptors but surprisingly not all receptors. This AI driven analysis of aGPCR structures provides a framework for exploring alternative receptor activation mechanisms. These models also offer valuable insights into receptor conformational changes and TL interactions, paving the way for the development of novel pharmacological tools and accelerating drug discovery efforts targeting this important receptor family.

BACKGROUND: SR-B1 (scavenger receptor class B type 1)/LDLR (low-density lipoprotein receptor) double knockout mice fed a high-fat, high-cholesterol diet containing cholate exhibit coronary artery disease characterized by occlusive coronary artery atherosclerosis, platelet accumulation in coronary arteries, and myocardial fibrosis. Platelets are involved in atherosclerosis development, and PAR (protease-activated receptor) 4 has a prominent role in platelet function in mice. However, the role of PAR4 on coronary artery disease in mice has not been tested. METHODS: We tested the effects of a PAR4 inhibitory pepducin (RAG8) on diet-induced aortic sinus and coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis in SR-B1/LDLR double knockout mice. SR-B1/LDLR double knockout mice were fed a high-fat, high-cholesterol diet containing cholate and injected daily with 20 mg/kg of either the RAG8 pepducin or a control reverse-sequence pepducin (SRQ8) for 20 days. RESULTS: Platelets from the RAG8-treated mice exhibited reduced thrombin and PAR4 agonist peptide–mediated activation compared with those from control SRQ8-treated mice when tested ex vivo. Although aortic sinus atherosclerosis levels did not differ, RAG8-treated mice exhibited reduced coronary artery atherosclerosis, reduced platelet accumulation in atherosclerotic coronary arteries, and reduced myocardial fibrosis. These protective effects were not accompanied by changes in circulating lipids, inflammatory cytokines, or immune cells. However, RAG8-treated mice exhibited reduced VCAM-1 (vascular cell adhesion molecule 1) protein levels in nonatherosclerotic coronary artery cross sections and reduced leukocyte accumulation in atherosclerotic coronary artery cross sections compared with those from SRQ8-treated mice. CONCLUSIONS: The PAR4 inhibitory RAG8 pepducin reduced coronary artery atherosclerosis and myocardial fibrosis in SR-B1/LDLR double knockout mice fed a high-fat, high-cholesterol diet containing cholate. Furthermore, RAG8 reduced VCAM-1 in nonatherosclerotic coronary arteries and reduced leukocyte and platelet accumulation in atherosclerotic coronary arteries. These findings identify PAR4 as an attractive target in reducing coronary artery disease development, and the use of RAG8 may potentially be beneficial in cardiovascular disease.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.16177. G protein‐coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.