Targeting the Atypical Chemokine Receptor ACKR3/CXCR7: Phase 1 - Phage Display Peptide Identification and Characterization.

One of the greatest challenges in fighting cancer is cell targeting and biomarker selection. The Atypical Chemokine Receptor ACKR3/CXCR7 is expressed on many cancer cell types, including breast cancer and glioblastoma, and binds the endogenous ligands SDF1/CXCL12 and ITAC/CXCL11. A 20 amino acid region of the ACKR3/CXCR7 N-terminus was synthesized and targeted with the NEB PhD-7 Phage Display Peptide Library. Twenty-nine phages were isolated and heptapeptide inserts sequenced; of these, 23 sequences were unique. A 3D molecular model was created for the ACKR3/CXCR7 N-terminus by mutating the corresponding region of the crystal structure of CXCR4 with bound SDF1/CXCL12. A ClustalW alignment was performed on each peptide sequence using the entire SDF1/CXCL12 sequence as the template. The 23-peptide sequences showed similarity to three distinct regions of the SDF1/CXCL12 molecule. A 3D molecular model was made for each of the phage peptide inserts to visually identify potential areas of steric interference of peptides that simulated CXCL12 regions not in contact with the receptor's Nterminus. An ELISA analysis of the relative binding affinity between the peptides identified 9 peptides with statistically significant results. The candidate pool of 9 peptides was further reduced to 3 peptides based on their affinity for the targeted N-terminus region peptide versus no target peptide present or a scrambled negative control peptide. The results clearly show the Phage Display protocol can be used to target a synthesized region of the ACKR3/CXCR7 N-terminus. The 3 peptides chosen, P20, P3, and P9, will be the basis for further targeting studies.

A systematic screen for dominant second-site modifiers of Merlin/NF2 phenotypes reveals an interaction with blistered/DSRF and scribbler.

Merlin, the Drosophila homologue of the human tumor suppressor gene Neurofibromatosis 2 (NF2), is required for the regulation of cell proliferation and differentiation. To better understand the cellular functions of the NF2 gene product, Merlin, recent work has concentrated on identifying proteins with which it interacts either physically or functionally. In this article, we describe genetic screens designed to isolate second-site modifiers of Merlin phenotypes from which we have identified five multiallelic complementation groups that modify both loss-of-function and dominant-negative Merlin phenotypes. Three of these groups, Group IIa/scribbler (also known as brakeless), Group IIc/blistered, and Group IId/net, are known genes, while two appear to be novel. In addition, two genes, Group IIa/scribbler and Group IIc/blistered, alter Merlin subcellular localization in epithelial and neuronal tissues, suggesting that they regulate Merlin trafficking or function. Furthermore, we show that mutations in scribbler and blistered display second-site noncomplementation with one another. These results suggest that Merlin, blistered, and scribbler function together in a common pathway to regulate Drosophila wing epithelial development.

The neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation.

Neurofibromatosis-2 is an inherited disorder characterized by the development of benign schwannomas and other Schwann-cell-derived tumors associated with the central nervous system. The Neurofibromatosis-2 tumor suppressor gene encodes Merlin, a member of the Protein 4.1 superfamily most closely related to Ezrin, Radixin and Moesin. This discovery suggested a novel function for Protein 4.1 family members in the regulation of cell proliferation; proteins in this family were previously thought to function primarily to link transmembrane proteins to underlying cortical actin. To understand the basic cellular functions of Merlin, we are investigating a Drosophila Neurofibromatosis-2 homologue, Merlin. Loss of Merlin function in Drosophila results in hyperplasia of the affected tissue without significant disruptions in differentiation. Similar phenotypes have been observed for mutations in another Protein 4.1 superfamily member in Drosophila, expanded. Because of the phenotypic and structural similarities between Merlin and expanded, we asked whether Merlin and Expanded function together to regulate cell proliferation. In this study, we demonstrate that recessive loss of function of either Merlin or expanded can dominantly enhance the phenotypes associated with mutations in the other. Consistent with this genetic interaction, we determined that Merlin and Expanded colocalize in Drosophila tissues and cells, and physically interact through a conserved N-terminal region of Expanded, characteristic of the Protein 4.1 family, and the C-terminal domain of Merlin. Loss of function of both Merlin and expanded in clones revealed that these proteins function to regulate differentiation in addition to proliferation in Drosophila. Further genetic analyses suggest a role for Merlin and Expanded specifically in Decapentaplegic-mediated differentiation events. These results indicate that Merlin and Expanded function together to regulate proliferation and differentiation, and have implications for understanding the functions of other Protein 4.1 superfamily members.

Structural analysis of Drosophila merlin reveals functional domains important for growth control and subcellular localization.

Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). NF2 is a dominantly inherited disease characterized by the formation of bilateral acoustic schwannomas and other benign tumors associated with the central nervous system. To understand its cellular functions, we are studying a Merlin homologue in Drosophila. As is the case for NF2 tumors, Drosophila cells lacking Merlin function overproliferate relative to their neighbors. Using in vitro mutagenesis, we define functional domains within Merlin required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane- associated NH2-terminal 350 amino acids of Merlin. Removal of a seven-amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. In addition, we provide evidence that the COOH-terminal region of Merlin has a negative regulatory role, as has been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene.

Isolation of mutations in the Drosophila homologues of the human Neurofibromatosis 2 and yeast CDC42 genes using a simple and efficient reverse-genetic method.

Reverse genetic analysis in Drosophila has been greatly aided by a growing collection of lethal P transposable element insertions that provide molecular tags for the identification of essential genetic loci. However, because the screens performed to date primarily have generated autosomal P-element insertions, this collection has not been as useful for performing reverse genetic analysis of X-linked genes. We have designed a reverse genetic screen that takes advantage of the hemizygosity of the X chromosome in males together with a cosmid-based transgene that serves as an autosomally linked duplication of a small region of the X chromosome. The efficacy and efficiency of this method is demonstrated by the isolation of mutations in Drosophila homologues of two well-studied genes, the human Neurofibromatosis 2 tumor suppressor and the yeast CDC42 gene. The method we describe should be of general utility for the isolation of mutations in other X-linked genes, and should also provide an efficient method for the isolation of new allcles of existing X-linked or autosomal mutations in Drosophila.