Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and long-term medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical and biochemical assays confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (KD ∼ 300 nM), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSV-G mediated infection, of ACE2 expressing human cells with an IC50 of 2.4± 0.1 μM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as novel agents to prevent SARS-CoV-2 infection.

C-reactive protein (CRP) is normally synthesized by hepatocytes at relatively low rates and is retained within the endoplasmic reticulum (ER) via interaction with two carboxylesterases (termed gp60a and gp60b), which themselves are restricted to the ER by their COOH-terminal retention signals (HIEL and HTEL).During the acute phase response, an increase in CRP synthesis is accompanied by a decrease in its ER retention as a result of a decrease in the CRP binding affinity of gp60b. Our previous data indicated that the esterase active site, the CRP binding site, and the ER retention signal are functionally distinct. In the present studies, we have identified CRP-binding peptides produced by proteolytic cleavage of gp60a. The sequence shared by two CRP-binding peptides indicated that the CRP binding site of gp60a is contained within residues 477-499. These results were confirmed by expression of cDNAs coding for gp60a and b as bacterial fusion proteins. Fusion proteins containing the complete esterase COOH terminus bound CRP, whereas those truncated at residue 477 (or the homologous site in gp60b) did not. Based on the known crystal structures of three homologous hydrolases, the putative CRP-binding site of the gp60s is located on the surface and is physically distant from the esterase active site and the COOH-terminal ER retention signal. C-reactive protein (CRP)1 is a plasma protein whose rate of synthesis by hepatocytes may increase by several hundred-fold or more during the systemic acute phase response to tissue injury (1). CRP is a homopentamer composed of 24-kDa nonglycosylated subunits (2) that assemble within the endoplasmic reticulum (ER) shortly after their synthesis (3). Under normal physiologic conditions CRP is synthesized at relatively low rates and is largely retained within the ER without being degraded: the half-time for exit of pulse-labeled CRP from the ER in hepatocytes isolated from normal rabbits was found to be in excess of 18 h (3, 4), whereas CRP was efficiently secreted with a half-time of only 75 min in cells isolated from animals stimulated in vivo to undergo the acute phase response (4). This differential retention of CRP within the cell represents a novel mechanism capable of regulating the intracellular sorting of a secretory protein.We have previously shown that retention of CRP within the ER is the result of its association with two 60-kDa glycosylated microsomal carboxylesterases (5, 6), which we have termed gp60a and gp60b (7). gp60a is an abundant ER protein with a relatively low affinity for CRP (K d ϭ 120 nM), whereas gp60b is a less abundant protein, but with a higher binding affinity for CRP (K d ϭ 1 nM) (3, 7). These esterases are restricted to the lumen of the ER by virtue of their carboxyl-terminal sequences of HIEL and HTEL (5, 6), which have been shown to be effective ER retention signals (8, 9), presumably being recognized by the KDEL retrieval pathway (10). Thus, we have proposed that the retention of CRP within the ER is mediated by its interaction with gp60a and b, w...

Plexins receive guidance cues from semaphorin ligands and transmit their signal through the plasma membrane. This family of proteins is unique amongst single-pass transmembrane receptors as their intracellular regions interact directly with several small GTPases, which regulate cytoskeletal dynamics and cell adhesion. Here, we characterize the GTPase Activating Protein (GAP) function of Plexin-B1 and find that a cooperative GAP activity towards the substrate GTPase, Rap1b, is associated with the N-terminal Juxtamembrane region of Plexin-B1. Importantly, we unveil an activation mechanism of Plexin-B1 by identifying a novel functional loop which partially blocks Rap1b entry to the plexin GAP domain. Consistent with the concept of allokairy developed for other systems, Plexin-B activity is increased by an apparent substrate mediated cooperative effect. Simulations and mutagenesis suggest the repositioned JM conformation is stabilized by the new activation switch loop when the active site is occupied, giving rise to faster enzymatic turnover and cooperative behavior. The biological implications, essentially of a threshold behavior for cell migration are discussed..