It was, until recently, accepted that the two classes of acetylcholine (ACh) receptors are distinct in an important sense: muscarinic ACh receptors signal via heterotrimeric GTP binding proteins (G proteins), whereas nicotinic ACh receptors (nAChRs) open to allow flux of Na+, Ca2+, and K+ ions into the cell after activation. Here we present evidence of direct coupling between G proteins and nAChRs in neurons. Based on proteomic, biophysical, and functional evidence, we hypothesize that binding to G proteins modulates the activity and signaling of nAChRs in cells. It is important to note that while this hypothesis is new for the nAChR, it is consistent with known interactions between G proteins and structurally related ligand-gated ion channels. Therefore, it underscores an evolutionarily conserved metabotropic mechanism of G protein signaling via nAChR channels.

Receptor function is dependent on interaction with various intracellular proteins that ensure the localization and signaling of the receptor. While a number of approaches have been optimized for the isolation, purification, and proteomic characterization of receptor–protein interaction networks (interactomes) in cells, the capture of receptor interactomes and their dynamic properties remains a challenge. In particular, the study of interactome components that bind to the receptor with low affinity or can rapidly dissociate from the macromolecular complex is difficult. Here we describe how chemical crosslinking (CC) can aid in the isolation and proteomic analysis of receptor–protein interactions. The addition of CC to standard affinity purification and mass spectrometry protocols boosts the power of protein capture within the proteomic assay and enables the identification of specific binding partners under various cellular and receptor states. The utility of CC in receptor interactome studies is highlighted for the nicotinic acetylcholine receptor as well as several other receptor types. A better understanding of receptors and their interactions with proteins spearheads molecular biology, informs an integral part of bench medicine which helps in drug development, drug action, and understanding the pathophysiology of disease.

Background: The HER2 antibody drug conjugate (ADC) fam-trastuzumab deruxtecan-nxki (T-DXd) significantly improves outcomes over standard chemotherapy in pts with HER2-LOW (IHC 1+ and IHC 2+ FISH-) metastatic breast cancer (MBC). Data from the DAISY (NCT04132960) trial in pts with HER2 IHC 0 or “ultra low” MBC revealed median progression-free survival (PFS) of 4.2 mos vs 6.7 mos in HER2-LOW and 11.1 mos in HER2 3+ pts (Diéras V, et al. Cancer Res. 2022;82(4_Suppl):PD8-02). There is an urgent need to develop methods to accurately discern HER2 LOW expression versus HER2 IHC 0/ultra-low expression so appropriate pts may receive T-DXd. We have developed a quantitative HER2 assay using reverse phase protein array (RPPA) technology with laser capture microdissection (LCM) enrichment of tumor epithelium that measures HER2 expression over a 65-fold dynamic range in HER2 IHC 0 to 3+ breast cancers. In I-SPY 1 and 2 trials we found that RPPA-assessed quantitative HER2 and activated/phosphorylated HER2 expression predicted for pathologic complete response in pts with HER2 IHC 0, HER2-LOW and HER2 3+ disease with various HER2 and other targeted agents. We then developed and validated the first CLIA/CAP-accredited quantitative HER2 protein expression and activation assay, and now explore quantitative HER2 expression in HER2 IHC 0/ultra low disease. Given the recent approval of sacituzumab govitecan-hziy in TNBC and the results of the TROPiCS-02 (NCT03901339), we also evaluated quantitative RPPA-based TROP-2 expression levels in HER2 IHC 0 breast cancer. Methods: LCM-enriched tumor epithelium was obtained from freshly cut FFPE core needle and resected breast cancer samples from 175 pts with pathology-determined HER2 IHC 0 status (N=68 ER+ and N=107 ER-). Quantitative HER2 output is generated as a relative intensity unit (RU) that is interpolated to an internal calibrator curve and a population referent comprised of known HER2+ (IHC 3+ and 2+/FISH+), HER2-LOW and HER2 IHC 0/ultra-low tumors to generate population-based cut-points as a referent. RPPA-based quantitative HER2 expression are reported as one of four levels of expression across the HER2 dynamic range observed: “non/extremely low”, “modest“, “moderate”, and “high”. Quantitative TROP2 protein expression levels were also measured for each case by the RPPA assay on the same lysate using the same adjectival determinants of relative expression as HER2. Results: For the HER2 IHC 0 ER+ cohort, we observed HER2 protein expression over a 25-fold dynamic range and that 57% (N=39) had HER2 non/extremely low, 37% (N=25) modest, and 4 (6%) moderate/high HER2 expression by RPPA. For the HER2 IHC 0 ER- cohort, we observed HER2 protein expression over a 15-fold dynamic range and that 71% (N=76) had non/extremely low, and 31% (N=29%) modest HER2 expression. We observed TROP-2 protein expression over a 35-fold dynamic range across all tumors measured, and it was found to be at least modestly expressed in 95% (37/39) ER+ and 95% (72/76) ER- of the IHC 0/ultra-low tumors that were also found to be HER2 non/extremely low by RPPA. Conclusions: LCM-RPPA quantitative assessment of HER2 expression showed that nearly 40% of ER+ IHC 0 and 30% of ER- IHC 0 “ultra-low” breast cancers actually had modest to moderate HER2 expression. Interestingly, this frequency approximates the response rate of 30% seen with T-DXd in HER2 IHC 0 pts in the DAISY trial. Quantitatively measured TROP2 is at least modestly expressed in the vast majority of RPPA-assessed HER2 IHC 0 cancers regardless of ER status, making a TROP2-directed ADC an attractive therapeutic option for these pts. If validated in larger studies, LCM-RPPA-based HER2 expression could provide a better understanding of the potential for therapeutic efficacy with T-DXd in patients with HER2 IHC “ultra-low” disease, and may better define true ultra-low HER2 expression in HER2 IHC 0 tumor biopsies at baseline and refine the lower limit of the HER2-LOW designation. Citation Format: Emanuel F. Petricoin, Brian A. Corgiat, Joyce O’Shaughnessy, Patricia LoRusso, Kris Weinberg, Justin Davis, Chelsea Gawryletz. HER2-17 Novel Quantitative HER2 Assay for Determining Dynamic HER2 Expression in the HER2 IHC 0 “Ultra-Low” Setting: Implications for Precision Therapy in HER2- Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-17.

Laser capture microdissection (LCM) is a technique that allows procurement of an enriched cell population from a heterogeneous tissue sample under direct microscopic visualization. Fundamentally, laser capture microdissection consists of three main steps: (1) visualizing the desired cell population by microscopy, (2) melting a thermolabile polymer onto the desired cell populations using infrared laser energy to form a polymer-cell composite (capture method) or photovolatizing a region of tissue using ultraviolet laser energy (cutting method), and (3) removing the desired cell population from the heterogeneous tissue. In this chapter, we discuss the infrared capture method only. LCM technology is compatible with a wide range of downstream applications such as mass spectrometry, DNA genotyping and RNA transcript profiling, cDNA library generation, proteomics discovery, and signal pathway mapping. This chapter profiles the ArcturusXT laser capture microdissection instrument, using isolation of specific cortical lamina from nonhuman primate brain regions, and sample preparation methods for downstream proteomic applications.