The short third intracellular loop and cytoplasmic tail of bitter taste receptors provide functionally relevant GRK phosphorylation sites in TAS2R14

For most GPCRs, the third intracellular loops (IL3) and C-terminal tails (CT) are sites for GRK-mediated phosphorylation, leading to b-arrestin binding and agonist-specific desensitization. These regions of the G protein-coupled bitter taste receptors (TAS2Rs) are short compared to the superfamily, and their functional role is unclear. TAS2R14 expressed on human airway smooth muscle (HASM) cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated GST-fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences. In vitro, activated GRK2 phosphorylated both WT IL3 and WT CT proteins but not Ala-substituted forms. Next, TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A) and in both regions (IL/CT-10A) were expressed in HEK-293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the [Ca2+]i response compared to WT, indicating functional desensitization by GRK-phosphorylation is at residues in the CT. Short-term desensitization of TAS2R14 was blocked by GRK2 knockdown in HASM cells. Receptor:b-arrestin binding was absent with IL/CT-10A and CT-5A, but was also reduced in IL-5A, indicating a role for IL3 phosphorylation in the b-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired and these receptors failed to colocalize with early endosomes. These results show that agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and b-arrestin binding. However, b-arrestin function in the internalization and trafficking of the receptor requires cooperative GRK phosphorylation of IL3 and CT residues.

The Two SH2 Domains of SHP-2 Bridge Two PD-1 Molecules Resulting in SHP-2 Activation and PD-1-Mediated Inhibition

Programmed cell death-1 (PD-1) is a checkpoint inhibitory receptor member of the B7-CD28 family, which promotes peripheral tolerance and restrains anti-viral and anti-tumor immunity. Although PD-1 blockade leads to durable clinical responses in a fraction of patients, the majority of patients display only transient responses, emphasizing the need for better understanding of the mechanism of PD-1-mediated T cell inhibition. In contrast to other CD28 family family members, which form disulfide-linked homodimers, PD-1 lacks the juxtamembrane cysteine residue responsible for homodimer formation and has been shown to exist as a monomer. The cytoplasmic tail of PD-1 has one immunoreceptor tyrosine-based inhibitory motif (ITIM), centered on Y223 residue, and one immunoreceptor tyrosine-based switch motif (ITSM), centered on Y248 residue. Mutational studies have shown that PD-1-mediated inhibition relies on the interaction of the ITSM with SHP-2 but the mechanism by which PD-1 induces SHP-2 activation is unknown. In this study we sought to determine how PD-1: SHP-2 interaction leads to inhibition of T-cell responses. SHP-2 contains a phosphatase (PTP) domain and two SH2 domains, N-SH2 and C-SH2. To determine whether PD-1 selectively interacts with a specific SH2 domain of SHP-2, we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Surprisingly, pull-down assays with GST-fusion proteins using lysates from primary human T cells or Jurkat T cell line revealed that PD-1 interacted with SHP-2 via both SH2 domains of SHP-2. Biacore assays confirmed that phosphorylated ITSM Y248 interacted with SHP-2 N-SH2 and C-SH2. To determine whether one of the SHP-2 SH2 domains might preferentially interact with PD-1 in live cells, we mutagenized the functional sites of the N-SH2 or C-SH2 domain at arginine R32 and R138, respectively, and transfected COS cells with cDNA of SHP-2 wild type or each SHP-2 SH2 mutant, together with PD-1 and the TCR proximal kinase Fyn, required for PD-1 phosphorylation and interaction with SHP-2. Immunoprecipitation and immunoblot assays showed that mutagenesis of either SH2 domain abrogated interaction of SHP-2 with PD-1 ITSM Y248, providing evidence that both SH2 domains of SHP-2 are involved in the interaction with PD-1. Because each PD-1 molecule has only one ITSM, these results indicate that SHP-2 interacts via its two SH2 domains with two PD-1 molecules. Assessment of PD-1: PD-1 dimer formation in live cells by split luciferase complementation, using NanoBiT proximity assays, showed that upon PD-1 phosphorylation, PD-1: PD1 interaction occurs only in the presence of SHP-2 with intact N-SH2 and C-SH2 domains. The SH2 domains of SHP-2 have a crucial and distinct role in regulating SHP-2 PTPase activity. In the absence of a tyrosine-phosphorylated binding ligand, N-SH2 is bound to the PTP domain leading to an auto-inhibitory closed conformation that blocks the PTP active site. Phosphorylation of Y542 in the SHP-2 C-terminus tail leads to intramolecular interaction of Y542 with the N-SH2 domain to relieve binding to PTP domain. Similarly, intermolecular interaction of the N-SH2 domain with specific phosphorylated ligands disrupts its PTP recognition surface reversing the auto-inhibitory conformation and activates the phosphatase. We determined that monomeric PD-1 ITIM-pY223 or ITSM-pY248 peptides did not activate the PTP. A dimeric phosphopeptide generated by covalent joining of two PD-1 ITSM-pY248 phosphopeptides with a linker that matches the distance between the binding sites of the two SHP-2 SH2 domains induced rapid activation of SHP-2. In contrast, similarly designed dimeric peptides joining either two PD-1 ITIM-pY223 or ITIM-pY223-and-ITSM-pY248 phosphopeptides did not activate PTP activity of SHP-2. The ability of PD1 ITSM-pY248 to induce SHP-2 activation was correlated with inhibition of antigen-mediated IL-2 production, which was abrogated when Y248 was mutagenized to phenylalanine. Our results reveal the geometry of PD-1: SHP-2 interaction that leads to SHP-2 activation and have implications for the development of PD-1-binding compounds to selectively suppress T cell responses by dimerizing PD-1 or enhance T cell responses by disrupting PD-1 dimerization and SHP-2 activation. Disclosures Freeman: EMD-Serono: Patents & Royalties; Xios: Membership on an entity's Board of Directors or advisory committees; Origimed: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Patents & Royalties; Bristol-Myers-Squibb: Patents & Royalties; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees; Roche: Patents & Royalties; Dako: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Merck: Patents & Royalties; Novartis: Patents & Royalties.

Covalent and selective immobilization of GST fusion proteins with fluorophosphonate-based probes

Fluorophosphonate probes covalently immobilize proteins onto solid support by reacting with tyrosine 111 in the GST tag.

Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

ABSTRACTMycoplasma boviscauses a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected withM. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed inEscherichia colias glutathioneS-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity withM. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detectedM. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detectedM. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.

Distinct Roles Of PD-1 Itsm and ITIM In Regulating Interactions With SHP-2, ZAP-70 and Lck, and PD-1-Mediated Inhibitory Function

Programmed death (PD)-1 plays a prominent role in the induction and maintenance of peripheral tolerance. The biochemical mechanisms via which PD-1 mediates its inhibitory function remain poorly understood. The cytoplamsic tail of PD-1 contains two structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). It has been reported that SHP-2 tyrosine phosphatase constitutively interacts with PD-1 ITSM and is involved in PD-1-mediated inhibitory function. We sought to identify the nature of PD-1: SHP-2 interaction and to determine whether other TCR-proximal signaling molecules might interact with PD-1 cytoplasmic tail. SHP-2 has two SH2 domains (N-SH2 and C-SH2) and one PTP domain. To identify the region of SHP-2 that interacts with PD-1 we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Pull down assays with each GST-fusion protein using lysates from naive and activated primary human T cells revealed that PD-1 interacted with GST-SHP-2 fusion protein only after T cell activation along with simultaneous PD-1 ligation. This interaction was mediated selectively via the SH2 domains of SHP-2, indicating that PD-1 requires prior tyrosine phosphorylation in order to undergo interaction with SHP-2. To identify the mechanism of PD-1 tyrosine phosphorylation governing PD-1: SHP-2 interaction, we used COS cells to express PD-1 along with either empty vector, the TCR proximal tyrosine kinase Fyn, or a kinase inactive mutant of Fyn, followed by pull down with each SHP-2-GST fusion protein. No interaction between PD-1 and SHP-2-GST fusion proteins was detected in lysates from COS cells expressing empty vector or kinase inactive Fyn mutant. In contrast, in the presence of active Fyn, PD-1 underwent tyrosine phosphorylation and was able to interact with GST fusion proteins of SHP-2-FL, SHP-2-N-SH2, SHP-2-C-SH2 and SHP-2-ΔN-SH2 but not SHP-2-PTP, providing evidence that PD-1: SHP-2 interaction requires tyrosine phosphorylation of PD-1 by Src family kinases for subsequent SH2-mediated recruitment of SHP-2. To determine the structural and functional role of each individual tyrosine in the ITIM and the ITSM of PD-1 cytoplasmic tail in PD-1: SHP-2 interaction in vivo, we used Jurkat T cells to express cDNA of either PD-1 wild type, PD-1 with the ITIM tyrosine mutated to phenylalanine (PD-1.Y223F), PD-1 with the ITSM tyrosine mutated to phenylalanine (PD-1.Y248F) or PD-1 with both ITIM and ITSM tyrosines mutated to phenylalanine (PD-1.Y223F/Y248F). After activation, PD-1 wild type underwent tyrosine phosphorylation and developed a robust interaction with SHP-2. PD-1.Y223F retained the ability to undergo interaction with SHP-2 after activation, whereas PD-1.Y248F and PD-1.Y223F/Y248F were unable to interact with SHP-2. We examined whether the PD-1 cytopasmic phosphotyrosines might interact with other SH2 domain containing proteins with critical role in T cell activation. We determined that after T cell activation, PD-1 displayed interaction with ZAP-70 and with activated Lck as determined by PD-1 immunoprecipitation followed by immunoblot with antibodies specific for ZAP-70 and for the activation-specific phospho-LckY394. These interactions remained unaffected in T cells expressing PD-1.Y223F but were abrogated in T cells expressing PD-1.Y248F or PD-1.Y223F/Y248F indicating a mandatory role of phosphorylated ITSM but not ITIM for these associations. However, despite their distinct ability to mediate interactions of PD-1 with SHP-2, Lck and ZAP-70, both phosphorylated ITSM and ITIM had a mandatory role in the inhibitory effect of PD-1 on T cell activation. In T cells expressing either PD-1.Y223F or PD-1.Y248F, PD-1-mediated inhibition of IL-2 production was diminished by 50%, but was almost abrogated in T cells expressing the double mutant PD-1.Y223F/Y248F. Our results indicate that the cytoplasmic tail of PD-1 requires tyrosine phosphorylation in order to mediate phosphorylation-dependent interactions and inhibition on T cell activation. Although phosphorylation-dependent interactions of PD-1 with SHP-2, ZAP-70 and Lck involve Y248 in the ITSM, yet unidentified interactions of Y223 in the ITIM are mandatory for PD-1-mediated inhibitory function on T cell activation. Disclosures: Freeman: Boehringer-Ingelheim: Patents & Royalties; Bristol-Myers-Squibb/Medarex: Patents & Royalties; Roche/Genentech: Patents & Royalties; Merck: Patents & Royalties; EMD-Serrono: Patents & Royalties; Amplimmune: Patents & Royalties; CoStim Pharmaceuticals: Patents & Royalties; Costim Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.