Two C-terminal isoforms of Aplysia tachykinin-related peptide receptors exhibit phosphorylation-dependent and phosphorylation-independent desensitization mechanisms.

Diversity, a hallmark of G protein-coupled receptor (GPCR) signaling, partly stems from alternative splicing of a single gene generating more than one isoform for a receptor. Additionally, receptor responses to ligands can be attenuated by desensitization upon prolonged or repeated ligand exposure. Both phenomena have been demonstrated and exemplified by the deuterostome tachykinin signaling system, although the role of phosphorylation in desensitization remains a subject of debate. Here, we describe the signaling system for tachykinin-related peptides (TKRPs) in a protostome, mollusk Aplysia. We cloned the Aplysia TKRP precursor, which encodes three TKRPs (apTKRP-1, apTKRP-2a, and apTKRP-2b) containing the FXGXR-amide motif. In situ hybridization and immunohistochemistry showed predominant expression of TKRP mRNA and peptide in the cerebral ganglia. TKRPs and their posttranslational modifications were observed in extracts of central nervous system ganglia using mass spectrometry. We identified two Aplysia TKRP receptors (apTKRPRs), named apTKRPR-A and apTKRPR-B. These receptors are two isoforms generated through alternative splicing of the same gene and differ only in their intracellular C termini. Structure-activity relationship analysis of apTKRP-2b revealed that both C-terminal amidation and conserved residues of the ligand are critical for receptor activation. C-terminal truncates and mutants of apTKRPRs suggested that there is a C-terminal phosphorylation-independent desensitization for both receptors. Moreover, apTKRPR-B also exhibits phosphorylation-dependent desensitization through the phosphorylation of C-terminal Ser/Thr residues. This comprehensive characterization of the Aplysia TKRP signaling system underscores the evolutionary conservation of the TKRP and TK signaling systems, while highlighting the intricacies of receptor regulation through alternative splicing and differential desensitization mechanisms.

Affinity-Driven Aryl Diazonium Labeling of Peptide Receptors on Living Cells.

Peptide-receptor interactions play critical roles in a wide variety of physiological processes. Methods to link bioactive peptides covalently to unmodified receptors on the surfaces of living cells are valuable for studying receptor signaling, dynamics, and trafficking and for identifying novel peptide-receptor interactions. Here, we utilize peptide analogues bearing deactivated aryl diazonium groups for the affinity-driven labeling of unmodified receptors. We demonstrate that aryl diazonium-bearing peptide analogues can covalently label receptors on the surface of living cells using both the neurotensin and the glucagon-like peptide 1 receptor systems. Receptor labeling occurs in the complex environment of the cell surface in a sequence-specific manner. We further demonstrate the utility of this covalent labeling approach for the visualization of peptide receptors by confocal fluorescence microscopy and for the enrichment and identification of labeled receptors by mass spectrometry-based proteomics. Aryl diazonium-based affinity-driven receptor labeling is attractive due to the high abundance of tyrosine and histidine residues susceptible to azo coupling in the peptide binding sites of receptors, the ease of incorporation of aryl diazonium groups into peptides, and the relatively small size of the aryl diazonium group. This approach should prove to be a powerful and relatively general method to study peptide-receptor interactions in cellular contexts.

Label-Free Quantitation of Endogenous Peptides.

Liquid chromatography-mass spectrometry (LC-MS)-based peptidomics methods allow for the detection and identification of many peptides in a complex biological mixture in an untargeted manner. Quantitative peptidomics approaches allow for comparisons of peptide abundance between different samples, allowing one to draw conclusions about peptide differences as a function of experimental treatment or physiology. While stable isotope labeling is a powerful approach for quantitative proteomics and peptidomics, advances in mass spectrometry instrumentation and analysis tools have allowed label-free methods to gain popularity in recent years. In a general label-free quantitative peptidomics experiment, peak intensity information for each peptide is compared across multiple LC-MS runs. Here, we outline a general approach for label-free quantitative peptidomics experiments, including steps for sample preparation, LC-MS data acquisition, data processing, and statistical analysis. Special attention is paid to address run-to-run variability, which can lead to several major problems in label-free experiments. Overall, our method provides researchers with a framework for the development of their own quantitative peptidomics workflows applicable to quantitation of peptides from a wide variety of different biological sources.

Nontargeted Identification of D-Amino Acid-Containing Peptides Through Enzymatic Screening, Chiral Amino Acid Analysis, and LC-MS.

D-amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide's mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a nontargeted manner.

Proteomic Identification of Seasonally Expressed Proteins Contributing to Heart Function and the Avoidance of Skeletal Muscle Disuse Atrophy in a Hibernating Mammal.

Hibernation in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) takes place over 4-6 months and is characterized by multiday bouts of hypothermic torpor (5-7 °C core body temperature) that are regularly interrupted every 1-2 weeks by brief (12-24 h) normothermic active periods called interbout arousals. Our goal was to gain insight into the molecular mechanisms that underlie the hibernator's ability to preserve heart function and avoid the deleterious effects of skeletal muscle disuse atrophy over prolonged periods of inactivity, starvation, and near-freezing body temperatures. To achieve this goal, we performed organelle enrichment of heart and skeletal muscle at five seasonal time points followed by LC-MS-based label-free quantitative proteomics. In both organs, we saw an increase in the levels of many proteins as ground squirrels transition from an active state to a prehibernation state in the fall. Interestingly, seasonal abundance patterns identified DHRS7C, SRL, TRIM72, RTN2, and MPZ as potential protein candidates for mitigating disuse atrophy in skeletal muscle, and ex vivo contractile mechanics analysis revealed no deleterious effects in the ground squirrel's muscles despite prolonged sedentary activity. Overall, an increased understanding of protein abundance in hibernators may enable novel therapeutic strategies to treat muscle disuse atrophy and heart disease in humans.