The Src homology 2 (SH2) domain has a special role as one of the cornerstone examples of a "modular" domain. The interactions of this domain are very well-conserved, and have long been described as a bidentate, or "two-pronged plug" interaction between the domain and a phosphotyrosine (pTyr) peptide. Recent work has, however, highlighted unusual features of the SH2 domain that illustrate a greater diversity than was previously appreciated. In this review we discuss some of the novel and unusual characteristics across the SH2 family, including unusual peptide binding pockets, multiple pTyr recognition sites, recognition sites for unphosphorylated peptides, and recently identified variability in the conserved FLVR motif.

The Rho and Ras pathways play vital roles in cell growth, division and motility. Cross-talk between the pathways amplifies their roles in cell proliferation and motility and its dysregulation is involved in disease pathogenesis. One important interaction for cross-talk occurs between p120RasGAP (RASA1), a GTPase activating protein (GAP) for Ras, and p190RhoGAP (p190RhoGAP-A, ARHGAP35), a GAP for Rho. The binding of these proteins is primarily mediated by two SH2 domains within p120RasGAP engaging phosphorylated tyrosines of p190RhoGAP, of which the best studied is pTyr-1105. To better understand the interaction between p120RasGAP and p190RhoGAP, we determined the 1.75 Å X-ray crystal structure of the N-terminal SH2 domain of p120RasGAP in the unliganded form, and its 1.6 Å co-crystal structure in complex with a synthesized phosphotyrosine peptide, EEENI(p-Tyr)SVPHDST, corresponding to residues 1100–1112 of p190RhoGAP. We find that the N-terminal SH2 domain of p120RhoGAP has the characteristic SH2 fold encompassing a central beta-sheet flanked by two alpha-helices, and that peptide binding stabilizes specific conformations of the βE-βF loop and arginine residues R212 and R231. Site-directed mutagenesis and native gel shifts confirm phosphotyrosine binding through the conserved FLVR motif arginine residue R207, and isothermal titration calorimetry finds a dissociation constant of 0.3 ± 0.1 μM between the phosphopeptide and SH2 domain. These results demonstrate that the major interaction between two important GAP proteins, p120RasGAP and p190RhoGAP, is mediated by a canonical SH2-pTyr interaction.

The Src homology 2 (SH2) domain has a highly conserved architecture that recognizes linear phosphotyrosine motifs and is present in a wide range of signaling pathways across different evolutionary taxa. A hallmark of SH2 domains is the arginine residue in the conserved “FLVR” motif that forms a direct salt bridge with bound phosphotyrosine. Here, we solve the X-ray crystal structures of the C-terminal SH2 domain of p120RasGAP (RASA1) in its apo and peptide-bound form. We find that the arginine residue in the FLVR motif does not directly contact pTyr-1087 of a bound phosphopeptide derived from p190RhoGAP; rather, it makes an intramolecular salt bridge to an aspartic acid. Unexpectedly, coordination of phosphotyrosine is achieved by a modified binding pocket which appears early in evolution. Using isothermal titration calorimetry, we find that substitution of the FLVR arginine R377A does not cause a significant loss of phosphopeptide binding, but rather a tandem substitution of R398A (SH2 position βD4) and K400A (SH2 position βD6) is required to disrupt the binding. These results indicate a hitherto unrecognized diversity in SH2 domain interactions with phosphotyrosine, and classify the C-terminal SH2 domain of p120RasGAP as “FLVR-unique.”

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ImportanceAdvances in treatment of metastatic breast cancer (MBC) led to changes in clinical practice and treatment costs in the US over the past decade. There is limited information on current MBC treatment sequences and associated costs by MBC subtype in the US.ObjectivesTo identify treatment patterns by MBC subtype and associated anticancer and supportive drug costs from health care sector and Medicare perspectives.Design, Setting, and ParticipantsThis economic evaluation analyzed data of patients with MBC obtained from the nationwide Flatiron Health database, an electronic health record–derived, deidentified database with data from community and academic practices across the US from 2011 to 2021. Participants included women aged at least 18 years diagnosed with MBC, who had at least 6 months of follow-up data, known hormone receptor (HR) and human epidermal growth factor receptor 2 (ERBB2) receptor status, and at least 1 documented line of therapy. Patients with documented receipt of clinical study drugs were excluded. Data were analyzed from June 2021 to May 2022.Main Outcomes and MeasuresOutcomes of interest were frequency of different drug regimens received as a line of therapy by subtype for the first 5 lines and mean medical costs of documented anticancer treatment and supportive care drugs per patient by MBC subtype and years since metastatic diagnosis, indexed to 2021 US dollars.ResultsAmong 15 215 patients (10 171 patients [66.85%] with HR-positive and ERBB2-negative MBC; 2785 patients [18.30%] with HR-positive and ERBB2-positive MBC; 802 patients [5.27%] with HR-negative and ERBB2-positive MBC; 1457 patients [9.58%] with triple-negative breast cancer [TNBC]) who met eligibility criteria, 1777 (11.68%) were African American, 363 (2.39%) were Asian, and 9800 (64.41%) were White; the median (range) age was 64 (21-84) years. The mean total per-patient treatment and supportive care drug cost using publicly available Medicare prices was $334 812 for patients with HR-positive and ERBB2-positive MBC, $284 609 for patients with HR-negative and ERBB2-positive MBC, $104 774 for patients with HR-positive and ERBB2-negative MBC, and $54 355 for patients with TNBC. From 2011 to 2019 (most recent complete year 1 data are for patients diagnosed in 2019), annual costs in year 1 increased from $12 986 to $80 563 for ERBB2-negative and HR-positive MBC, $99 997 to $156 712 for ERBB2-positive and HR-positive MBC, and $31 397 to $53 775 for TNBC.Conclusions and RelevanceThis economic evaluation found that drug costs related to MBC treatment increased between 2011 and 2021 and differed by tumor subtype. These findings suggest the growing financial burden of MBC treatment in the US and highlights the importance of performing more accurate cost-effectiveness analysis of novel adjuvant therapies that aim to reduce metastatic recurrence rates for early-stage breast cancer.