Detection of CXCR2 Cytokine Receptor Surface Expression Using Immunofluorescence

Lam, Clarissa; Pavel, Mahmud Arif; Kashyap, Parul; Salehi-Najafabadi, Zahra; Valentino, Victoria; Yu, Yong

doi:10.1007/978-1-4939-0928-5_17

Cited by 5 publications

(5 citation statements)

References 8 publications

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“…The expression of the recombinant proteins on the extracellular surface of the plasma membrane was then confirmed by the immunofluorescence experiment (37). When either TRPP2 S1-S2 and PKD1 S6-S7 loops or TRPP3 S1-S2 and PKD1L3 S6-S7 loops were coexpressed in HEK 293T cells, we detected cell surface expression of both pairs of proteins (Fig.…”

Section: Dna Constructs and Cell Surface Expression Of The Trpp And Pmentioning

confidence: 65%

Extracellular Loops Are Essential for the Assembly and Function of Polycystin Receptor-Ion Channel Complexes

Salehi-Najafabadi¹,

Li²,

Valentino

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellPolycystin complexes, or TRPP-PKD complexes, made of transient receptor potential channel polycystin (TRPP) and polycystic kidney disease (PKD) proteins, play key roles in coupling extracellular stimuli with intracellular Ca 2؉ signals. For example, the TRPP2-PKD1 complex has a crucial function in renal physiology, with mutations in either protein causing autosomal dominant polycystic kidney disease. In contrast, the TRPP3-PKD1L3 complex responds to low pH and was proposed to be a sour taste receptor candidate. It has been shown previously that the protein partners interact via association of the C-terminal or transmembrane segments, with consequences for the assembly, surface expression, and function of the polycystin complexes. However, the roles of extracellular components, especially the loops that connect the transmembrane segments, in the assembly and function of the polycystin complex are largely unknown. Here, with an immunoprecipitation method, we found that extracellular loops between the first and second transmembrane segments of TRPP2 and TRPP3 associate with the extracellular loops between the sixth and seventh transmembrane segments of PKD1 and PKD1L3, respectively. Immunofluorescence and electrophysiology data further confirm that the associations between these loops are essential for the trafficking and function of the complexes. Interestingly, most of the extracellular loops are also found to be involved in homomeric assembly. Furthermore, autosomal dominant polycystic kidney disease-associated TRPP2 mutant T448K significantly weakened TRPP2 homomeric assembly but had no obvious effect on TRPP2-PKD1 heteromeric assembly. Our results demonstrate a crucial role of these functionally underexplored extracellular loops in the assembly and function of the polycystin complexes.

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Section: Dna Constructs and Cell Surface Expression Of The Trpp And Pmentioning

confidence: 65%

Extracellular Loops Are Essential for the Assembly and Function of Polycystin Receptor-Ion Channel Complexes

Salehi-Najafabadi¹,

Li²,

Valentino

et al. 2017

Journal of Biological Chemistry

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“…The most evidence of chemokine receptors endogenously expressed by cancer cells is provided for CXCR1, CXCR2, CXCR4, and CXCR7 but also other receptors such as CCR1, CCR2, CCR3, CCR5, CCR7, CCR9, CXCR3, CXCR6, CXCR5, CX3CR1 are involved in the multistep process of prostate cancer progression and metastasis [78,[87][88][89][90][91][92]. Mainly, the work on chemokines in prostate cancer has focused on not only the chemokines CXCL8, CXCL12, and CCL2 but also other chemokines such as CCL2, CCL4, CCL5, CCL9, CCL11, CCL18, CXCL1, CXCL2, CXCL5, CXCL6, CXCL12, and CXCL13 that appear to be produced by stromal cells, endothelial cells and human bone marrow endothelial cells, differentiated osteoblasts, and infiltrating leukocytes [78,[93][94][95][96][97][98][99].…”

Section: Chemokines and Prostate Cancermentioning

confidence: 99%

Inflammation and prostate cancer: friends or foe?

Taverna

Pedretti

et al. 2015

Inflamm. Res.

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There are growing evidences that chronic inflammation is involved in the regulation of cellular events in prostate carcinogenesis, including disruption of the immune response and regulation of the tumor microenvironment. This review discusses the role played by the innate and adaptive immune system in the local progression of prostate cancer, and the prognostic information that we can currently understand and exploit.

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“…Immunofluorescence of HEK 293T cells was done with a protocol described previously [26]. Mouse monoclonal anti-HA (BioLegend), anti-FLAG (Sigma), and secondary antibody Alexa Fluor 488 and Alexa Fluor 594 (Life Technologies) were used.…”

Section: Immunofluorescencementioning

confidence: 99%

A PKD1L3 splice variant in taste buds is not cleaved at the G protein-coupled receptor proteolytic site

Kashyap

Wang

et al. 2019

Biochemical and Biophysical Research Communications

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Mutations in polycystin proteins PKD1 and TRPP2 lead to autosomal dominant polycystic kidney disease. These two proteins form a receptor-ion channel complex on primary cilia. PKD1 undergoes an autoproteolysis at the N terminal G-protein-coupled receptor proteolytic site (GPS), which is essential for the function of PKD1. Whether GPS cleavage happens in other PKD proteins and its functional consequence has remained elusive. Here we studied the GPS cleavage of PKD1L3, a protein that associates with TRPP3 in taste cells and may play a role in sour taste. Our results show that PKD1L3 also undergoes GPS cleavage. Mutation at the GPS abolishes the cleavage, and the non-cleavable mutant does not traffic to the plasma membrane when associated with TRPP3. We also found that a splicing variant of PKD1L3, which was originally identified in taste buds, is not cleaved. Amino acids L708 and S709, which are missing in this splicing variant, are crucial for the GPS cleavage of PKD1L3 and the trafficking of the PKD1L3/TRPP3 complex. Our results gain insight into the molecular mechanism of the GPS cleavage of PKD1L3. The presence of the non-cleavable variant suggests the potential in vivo function of uncleaved PKD proteins.

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Detection of CXCR2 Cytokine Receptor Surface Expression Using Immunofluorescence

Cited by 5 publications

References 8 publications

Extracellular Loops Are Essential for the Assembly and Function of Polycystin Receptor-Ion Channel Complexes

Extracellular Loops Are Essential for the Assembly and Function of Polycystin Receptor-Ion Channel Complexes

Inflammation and prostate cancer: friends or foe?

A PKD1L3 splice variant in taste buds is not cleaved at the G protein-coupled receptor proteolytic site

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