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G protein-coupled receptors (GPCRs) are the largest family of human proteins. They have a common structure and, signaling through a much smaller set of G proteins, arrestins, and effectors, activate downstream pathways that often modulate hallmark mechanisms of cancer. Because there are many more GPCRs than effectors, mutations in different receptors could perturb signaling similarly so as to favor a tumor. We hypothesized that somatic mutations in tumor samples may not be enriched within a single gene but rather that cognate mutations with similar effects on GPCR function are distributed across many receptors. To test this possibility, we systematically aggregated somatic cancer mutations across class A GPCRs and found a nonrandom distribution of positions with variant amino acid residues. Individual cancer types were enriched for highly impactful, recurrent mutations at selected cognate positions of known functional motifs. We also discovered that no single receptor drives this pattern, but rather multiple receptors contain amino acid substitutions at a few cognate positions. Phenotypic characterization suggests these mutations induce perturbation of G protein activation and/or β-arrestin recruitment. These data suggest that recurrent impactful oncogenic mutations perturb different GPCRs to subvert signaling and promote tumor growth or survival. The possibility that multiple different GPCRs could moonlight as drivers or enablers of a given cancer through mutations located at cognate positions across GPCR paralogs opens a window into cancer mechanisms and potential approaches to therapeutics. [ABSTRACT FROM AUTHOR]

Leucine rich repeat transmembrane (LRRTM) proteins are synaptic adhesion molecules with roles in synapse formation and signaling. LRRTM4 transcripts were previously shown to be enriched in rod bipolar cells (BCs), secondary neurons of the retina that form synapses with rod photoreceptors. Using two different antibodies, LRRTM4 was found to reside primarily at rod BC dendritic tips, where it colocalized with the transduction channel protein, TRPM1. LRRTM4 was not detected at dendritic tips of ON‐cone BCs. Following somatic knockout of LRRTM4 in BCs by subretinal injection and electroporation of CRISPR/Cas9, LRRTM4 was abolished or reduced in the dendritic tips of transfected cells. Knockout cells had a normal complement of TRPM1 at their dendritic tips, while GPR179 accumulation was partially reduced. In experiments with heterologously expressed protein, the extracellular domain of LRRTM4 was found to engage in heparan‐sulfate dependent binding with pikachurin. These results implicate LRRTM4 in the GPR179‐pikachurin‐dystroglycan transsynaptic complex at rod synapses. [ABSTRACT FROM AUTHOR]

The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the μ1 protein, each complexed with three copies of the protector protein Σ3. Conformational changes in μ1 following the proteolytic removal of Σ3 lead to release of the myristoylated N-terminal cleavage fragment μ1N and ultimately to membrane penetration. The μ1N fragment forms pores in red blood cell (RBC) membranes. In this report, we describe the interaction of recombinant μ1 trimers and synthetic μ1N peptides with both RBCs and liposomes. The μ1 trimer mediates hemolysis and liposome disruption under conditions that promote the μ1 conformational change, and mutations that inhibit μ1 conformational change in the context of intact virus particles also prevent liposome disruption by particle-free μ1 trimer. Autolytic cleavage to form μ1N is required for hemolysis but not for liposome disruption. Pretreatment of RBCs with proteases rescues hemolysis activity, suggesting that μ1N cleavage is not required when steric barriers are removed. Synthetic myristoylated μ1N peptide forms size-selective pores in liposomes, as measured by fluorescence dequenching of labeled dextrans of different sizes. Addition of a C-terminal solubility tag to the peptide does not affect activity, but sequence substitution V13N or L36D reduces liposome disruption. These substitutions are in regions of alternating hydrophobic residues. Their locations, the presence of an N-terminal myristoyl group, and the full activity of a C-terminally extended peptide, along with circular dichroism data that indicate prevalence of β-strand secondary structure, suggest a model in which μ1N β-hairpins assemble in the membrane to form a β-barrel pore. [ABSTRACT FROM AUTHOR]

Synaptic transmission from photoreceptors to ON-bipolar cells (BCs) requires the postsynaptic metabotropic glutamate receptor mGluR6, located at BC dendritic tips. Binding of the neurotransmitter glutamate initiates G protein signaling that regulates the TRPM1 transduction channel. mGluR6 also interacts with presynaptic ELFN adhesion proteins, and these interactions are important for mGluR6 synaptic localization. The mechanisms of mGluR6 trafficking and synaptic targeting remain poorly understood. In this study, we investigated mGluR6 missense mutations from patients with congenital stationary night blindness (CSNB), which is associated with loss of synaptic transmission to ON-BCs. We found that multiple CSNB mutations in the extracellular ligand-binding domain of mGluR6 impart a trafficking defect leading to lack of complex N-glycosylation but efficient plasma membrane insertion, suggesting a Golgi bypass mechanism. These mutants fail to bind ELFN1, consistent with lack of a necessary modification normally acquired in the Golgi. The same mutants were mislocalized in bipolar cells, explaining the loss of function in CSNB. The results reveal a key role of Golgi trafficking in mGluR6 function, and suggest a role of the extracellular domain in Golgi sorting., (© 2024 Pindwarawala et al.)

Photoreceptor cells in the vertebrate retina have a highly compartmentalized morphology for efficient long-term phototransduction. Rhodopsin, the visual pigment in rod photoreceptors, is densely packaged into the rod outer segment sensory cilium and continuously renewed through essential synthesis and trafficking pathways housed in the rod inner segment. Despite the importance of this region for rod health and maintenance, the subcellular organization of rhodopsin and its trafficking regulators in the mammalian rod inner segment remain undefined. We used super-resolution fluorescence microscopy with optimized retinal immunolabeling techniques to perform a single molecule localization analysis of rhodopsin in the inner segments of mouse rods. We found that a significant fraction of rhodopsin molecules was localized at the plasma membrane in an even distribution along the entire length of the inner segment, where markers of transport vesicles also colocalized. Thus, our results collectively establish a model of rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway in mouse rod photoreceptors., Competing Interests: The authors declare no competing financial interests.

Heat-resistant mutants selected from infectious subvirion particles of mammalian reoviruses have determinative mutations in the major outer-capsid protein micro 1. Here we report the isolation and characterization of intragenic pseudoreversions of one such thermostabilizing mutation. From a plaque that had survived heat selection, a number of viruses with one shared mutation but different second-site mutations were isolated. The effect of the shared mutation alone or in combination with second-site mutations was examined using recoating genetics. The shared mutation, D371A, was found to confer (i) substantial thermostability, (ii) an infectivity defect that followed attachment but preceded viral protein synthesis, and (iii) resistance to micro 1 rearrangement in vitro, with an associated failure to lyse red blood cells. Three different second-site mutations were individually tested in combination with D371A and found to wholly or partially revert these phenotypes. Furthermore, when tested alone in recoated particles, each of these three second-site mutations conferred demonstrable thermolability. This and other evidence suggest that pseudoreversion of micro 1-based thermostabilization can occur by a general mechanism of micro 1-based thermolabilization, not requiring a specific compensatory mutation. The thermostabilizing mutation D371A as well as 9 of the 10 identified second-site mutations are located near contact regions between micro 1 trimers in the reovirus outer capsid. The availability of both thermostabilizing and thermolabilizing mutations in micro 1 should aid in defining the conformational rearrangements and mechanisms involved in membrane penetration during cell entry by this structurally complex nonenveloped animal virus.