The switch to distance learning as a result of the COVID-19 pandemic has required chemistry instructors to quickly adapt and innovate to provide remote instruction as effectively as possible. With minimal advance notice, developing and delivering online lecture materials that are engaging and laboratory activities that approximate a hands-on experience has certainly been an all-around challenge. Creating content that ensures the inclusion and success of all students, including those who are deaf and hard-of-hearing (D/HH), requires a great deal of consideration to be effective under any circumstances. To be sure, mainstream courses that are inclusive of D/HH learners necessitate, at the very least, the successful inclusion of access services (interpreters and captionists). However, in these abnormal times, an online instructional environment that accommodates D/HH students’ preferences and abilities and that is attentive to the unique social and emotional challenges that many of these students are experiencing must be established. Our campus provides both direct instruction and mainstream instructional environments for D/HH students, giving us unique perspectives on how to instruct and support students with the pandemic-forced switch to online learning. With this communication, we endeavor to share these observations and experiences with other chemistry faculty so that they may support D/HH students in their own classrooms and laboratories.

The pituitary receptor for thyrotropin-releasing hormone (TRH) is a calcium-mobilizing G protein-coupled receptor (GPCR) that signals through Gq/11, elevating calcium, and activating protein kinase C. TRH receptor signaling is quickly desensitized as a consequence of receptor phosphorylation, arrestin binding, and internalization. Following activation, TRH receptors are phosphorylated at multiple Ser/Thr residues in the cytoplasmic tail. Phosphorylation catalyzed by GPCR kinase 2 (GRK2) takes place rapidly, reaching a maximum within seconds. Arrestins bind to two phosphorylated regions, but only arrestin bound to the proximal region causes desensitization and internalization. Phosphorylation at Thr365 is critical for these responses. TRH receptors internalize in clathrin-coated vesicles with bound arrestin. Following endocytosis, vesicles containing phosphorylated TRH receptors soon merge with rab5-positive vesicles. Over approximately 20 min these form larger endosomes rich in rab4 and rab5, early sorting endosomes. After TRH is removed from the medium, dephosphorylated receptors start to accumulate in rab4-positive, rab5-negative recycling endosomes. The mechanisms responsible for sorting dephosphorylated receptors to recycling endosomes are unknown. TRH receptors from internal pools help repopulate the plasma membrane. Dephosphorylation of TRH receptors begins when TRH is removed from the medium regardless of receptor localization, although dephosphorylation is fastest when the receptor is on the plasma membrane. Protein phosphatase 1 is involved in dephosphorylation but the details of how the enzyme is targeted to the receptor remain obscure. It is likely that future studies will identify biased ligands for the TRH receptor, novel arrestin-dependent signaling pathways, mechanisms responsible for targeting kinases and phosphatases to the receptor, and principles governing receptor trafficking.

The ␣-factor receptor Ste2p is a G protein-coupled receptor (GPCR) expressed on the surface of MATa haploid cells of the yeast Saccharomyces cerevisiae. Binding of ␣-factor to Ste2p results in activation of a heterotrimeric G protein and of the pheromone response pathway. Functional interactions between ␣-factor receptors, such as dominant-negative effects and recessive behavior of constitutive and hypersensitive mutant receptors, have been reported previously. We show here that dominant-negative effects of mutant receptors persist over a wide range of ratios of the abundances of G protein to receptor and that such effects are not blocked by covalent fusion of G protein ␣ subunits to normal receptors. In addition, we detected dominant effects of mutant C-terminally truncated receptors, which had not been previously reported to act in a dominant manner. Furthermore, coexpression of C-terminally truncated receptors with constitutively active mutant receptors results in enhancement of constitutive signaling. Together with previous evidence for oligomerization of Ste2p receptors, these results are consistent with the idea that functional interactions between coexpressed receptors arise from physical interactions between them rather than from competition for limiting downstream components, such as G proteins.

The thyrotropin-releasing hormone (TRH) receptor undergoes rapid and extensive agonist-dependent phosphorylation attributable to G protein-coupled receptor (GPCR) kinases (GRKs), particularly GRK2. Like many GPCRs, the TRH receptor is predicted to form an amphipathic helix, helix 8, between the NPXXY motif at the cytoplasmic end of the seventh transmembrane domain and palmitoylation sites at Cys335 and Cys337. Mutation of all six lysine and arginine residues between the NPXXY and residue 340 to glutamine (6Q receptor) did not prevent the receptor from stimulating inositol phosphate turnover but almost completely prevented receptor phosphorylation in response to TRH. Phosphorylation at all sites in the cytoplasmic tail was inhibited. The phosphorylation defect was not reversed by long incubation times or high TRH concentrations. As expected for a phosphorylation-defective receptor, the 6Q-TRH receptor did not recruit arrestin, undergo the typical arrestin-dependent increase in agonist affinity, or internalize well. Lys326, directly before phenylalanine in the common GPCR motif NPXXY(X) 5-6 F(R/K), was critical for phosphorylation. The 6Q-TRH receptor was not phosphorylated effectively in cells overexpressing GRK2 or in in vitro kinase assays containing purified GRK2. Phosphorylation of the 6Q receptor was partially restored by coexpression of a receptor with an intact helix 8 but without phosphorylation sites. Phosphorylation was inhibited but not completely prevented by alanine substitution for cysteine palmitoylation sites. Positively charged amino acids in the proximal tail of the ␤2-adrenergic receptor were also important for GRK-dependent phosphorylation. The results indicate that positive residues in helix 8 of GPCRs are important for GRK-dependent phosphorylation.The type 1 TRH receptor is a GPCR expressed in the anterior pituitary gland. In response to TRH, the receptor activates G q/11 , which in turn stimulates phospholipase C. The resulting increases in inositol trisphosphate and diacylglycerol cause rapid release of intracellular calcium and activation of protein kinase C. Like many GPCRs, the TRH receptor contains a canonical NPXXY at the end of transmembrane 7 in a NPXXY(X) 5-6 F(R/K) motif (Mirzadegan et al., 2003;Okuno et al., 2005;Swift et al., 2006) and is predicted to form an amphipathic eighth helix with a positively charged face ending at a downstream pair of cysteine residues, where palmitoylation occurs (Du et al., 2005).When TRH binds, the receptor undergoes rapid and quantitative phosphorylation at multiple sites in the cytoplasmic tail Hinkle, 2005, 2008;Jones et al., 2007). Agonist-stimulated phosphorylation of the receptor drives arrestin binding and arrestin-dependent receptor desensitization and internalization. The receptor tail is not detectably phosphorylated before activation.Agonist-dependent phosphorylation of GPCRs is usually carried out by either GRKs that recognize the activated state of the receptor or downstream kinases that are activated by receptor signaling. Th...

Oligomerization of G protein-coupled receptors is commonly observed, but the functional significance of oligomerization for this diverse family of receptors remains poorly understood. We used bioluminescence resonance energy transfer (BRET) to examine oligomerization of Ste2p, a G protein-coupled receptor that serves as the receptor for the ␣-mating pheromone in the yeast Saccharomyces cerevisiae, under conditions where the functional effects of oligomerization could be examined. Consistent with previous results from fluorescence resonance energy transfer (Overton, M. C., and Blumer, K. J. (2000) Curr. Biol. 10, 341-344), we detected efficient energy transfer between Renilla luciferase and a modified green fluorescent protein individually fused to truncated ␣-factor receptors lacking the cytoplasmic C-terminal tail. In addition, the low background of the BRET system allowed detection of significant, but less efficient, energy transfer between full-length receptors. The reduced efficiency of energy transfer between full-length receptors does not appear to result from different levels of receptor expression. Instead, attachment of fluorescent reporter proteins to the full-length receptors appears to significantly increase the distance between reporters. Mutations that were previously reported to block dimerization of truncated ␣-factor receptors reduce but do not completely eliminate BRET transfer between receptors. Dominant negative effects of mutant alleles of ␣-factor receptors appear to be mediated by receptor oligomerization since these effects are abrogated by introduction of additional mutations that reduce oligomerization. We find that heterodimers of normal and dominant negative receptors are defective in their ability to signal. Thus, signal transduction by oligomeric receptors appears to be a cooperative process requiring an interaction between functional monomers. G protein-coupled receptors (GPCRs)3 comprise a large family of cellular receptors responsible for transducing signals from a wide variety of extracellular stimuli including peptides, neurotransmitters, hormones, and light. All GPCRs are transmembrane proteins consisting of an extracellular N-terminal domain, seven transmembrane ␣-helical segments, and a cytoplasmic C-terminal tail. A large body of evidence indicates that GPCRs form homo-and/or hetero-oligomeric complexes in cells (2-5). Although the implications of oligomerization for receptor function remain poorly understood, in some cases oligomerization is capable of affecting biogenesis and membrane targeting of receptors (6, 7). In addition, cooperation between different monomers appears to be responsible for mediating or modulating the signaling function of some GPCRs (8, 9).The possibility of artifactual aggregation (or dis-aggregation) of GPCRs during solubilization and extraction from the membranes makes it desirable to monitor the oligomeric state of receptors that are maintained in their native cellular membranes. Because detection of nonradiative energy transfer between fluoresc...