Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients

Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity

Loss of E-cadherin provides tolerance to centrosome amplification in epithelial cancer cells

Centrosome amplification is a common feature of human tumors. To survive, cancer cells cluster extra centrosomes during mitosis, avoiding the detrimental effects of multipolar divisions. However, it is unclear whether clustering requires adaptation or is inherent to all cells. Here, we show that cells have varied abilities to cluster extra centrosomes. Epithelial cells are innately inefficient at clustering even in the presence of HSET/KIFC1, which is essential but not sufficient to promote clustering. The presence of E-cadherin decreases cortical contractility during mitosis through a signaling cascade leading to multipolar divisions, and its knockout promotes clustering and survival of cells with multiple centrosomes. Cortical contractility restricts centrosome movement at a minimal distance required for HSET/KIFC1 to exert its function, highlighting a biphasic model for centrosome clustering. In breast cancer cell lines, increased levels of centrosome amplification are accompanied by efficient clustering and loss of E-cadherin, indicating that this is an important adaptation mechanism to centrosome amplification in cancer

Discoidin domain receptors: multitaskers for physiological and pathological processes

Discoidin domain receptors: multitaskers for physiological and pathological processe

Identifying a targeting factor for Rab27a

The Rab family of small GTPases regulate intracellular membrane trafficking pathways and each Rab GTPase localises to a discrete membrane compartment. The mechanism(s) by which this targeting is achieved remains to be elucidated. Previous work identified that the C‐terminal hypervariable domain and effector binding were not necessary for Rab27a targeting in melanocytes. However, GEF activity of the non‐redundant Rab27a GEF Rab3GEP (R3G) was required but not sufficient for Rab27a targeting. This led to the hypothesis of the involvement of a Rab27a targeting factor. A Rab27a mutant (Rab27aSF1/F4) had previously been characterised that maintained melanosomal targeting and GTP‐loading by R3G but bound to no known Rab27a effectors. Using this mutant, novel Rab27a interacting partners were identified and assessed for their possible role in Rab27a targeting. The current research identified ATP1a1 (the α1 subunit of Na+,K+‐ATPase) as a novel interacting partner for Rab27a in melanocytes, depletion of which resulted in perinuclear clustering of melanosomes, indicative of loss of Rab27a function. Depletion of ATP1a1 did not disrupt Rab27a membrane localisation but did reduce the levels of GTP‐bound Rab27a, comparable to what is seen following depletion of R3G. Crucially, depletion of ATP1a1 resulted in displacement of endogenous Rab27a from melanosomal membranes indicating a role in Rab27a targeting. Furthermore, ATP1a1 depletion disrupted the levels of other melanosomal proteins indicative of a disruption in melanogenesis. ATP1a1 is proposed to transiently associate with melanosomes and through its Na+,K+‐ATPase pump function regulate the lumenal pH, which indirectly regulates melanosome maturation. How the maturation of the lumenal environment is conveyed to Rab27a, and is subsequent recruitment to melanosomes remains to be clarified.EThOS - Electronic Theses Online ServiceGBUnited Kingdo