Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins, thereby playing an essential role in the regulation of many physiological processes. These interactions are important for both normal signal transduction and regulation of the tissue distribution of signaling molecules. In this study, we use surface plasmon resonance (SPR) to study interactions of HS and structurally related heparin with proteins in the Hedgehog signaling pathway. SPR analysis shows that heparin binds with different affinities to active fragments of the proteins Hedgehog (Hh), Interference Hedgehog (Ihog), Cam-related/Down-regulated by Oncogenes (CDO), and Sonic Hedgehog (Shh). Solution competition studies show that the minimum size of a heparin oligosaccharide capable of interacting with Ihog is larger than a tetrasaccharide and for interacting with Shh is larger than an octasaccharide. In comparison with heparin, Ihog and Shh exhibited a lower affinity for HS than for heparin, and CDO and Hh exhibit negligible binding to HS. This study clearly demonstrates Shh and Ihog are heparin and HS binding proteins and that both molecules preferentially bind heparin or HS having a high level of sulfation.

Damage to the CNS can cause a differential spatio-temporal release of multiple factors, such as nucleotides, ATP and UTP. The latter interact with neuronal and glial nucleotide receptors. The P2Y 2 nucleotide receptor (P2Y 2 R) has gained prominence as a modulator of gliotic responses after CNS injury. Still, the molecular mechanisms underlying these responses in glia are not fully understood. Membrane-raft microdomains, such as caveolae, and their constituent caveolins, modulate receptor signaling in astrocytes; yet, their role in P2Y 2 R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y 2 R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y 2 R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, colocalize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y 2 R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y 2 R and Cav-1, and shRNA knockdown of Cav-1 expression. Furthermore, shRNA knockdown of Cav-1 expression decreased nucleotide-induced increases in the intracellular Ca 2؉ concentration in 1321N1 and C6 glioma cells without altering TRAP-6 and carbachol Ca 2؉ responses. In addition, Cav-1 shRNA knockdown also decreased AKT phosphorylation and altered the kinetics of ERK1/2 activation in 1321N1 cells. Our findings strongly suggest that P2Y 2 R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y 2 R/Cav-1 interactions represent a novel target for controlling P2Y 2 R function after CNS injury.Neurodegenerative conditions are among the leading causes of death and disability in the United States and have dramatically increased in incidence during the last decade (1, 2). The P2 receptors for extracellular nucleotides have emerged as key modulators of the pathophysiology of neurodegeneration (3-6). G protein-coupled P2Y 2 nucleotide receptors (P2Y 2 Rs) 3 have been identified in both neurons and glia as mediators of pro-inflammatory responses, neurotransmission, apoptosis, proliferation, and cell migration (3-5, 7, 8). In addition, the P2Y 2 Rs have also gained prominence, due to their association with some types of neoplasms, spinal cord injury, and the enhancement of neuronal differentiation (7, 9 -15). Further insight into the spatio-temporal organization of the P2Y 2 R and its signaling cascades in astrocytic cells is required to expand our knowledge of their role in neurodegenerative diseases. In this context, evidence suggests that receptors and associated signaling molecules are not randomly distributed in plasma membranes but are localized in specialized membrane microdomains, namely membrane rafts (MRs), such as caveolae (Cav) (16 -20). MRs are specialized membrane domains enriched in cholesterol and g...

Damage to the CNS cells’ can cause a differential spatiotemporal release of multiple factors into the extracellular space. Among such factors, nucleotides, and their interaction with the P2Y2 nucleotide receptors (P2Y2Rs) have gained prominence as putative modulators of gliotic responses after CNS injury. Yet, the molecular mechanisms underlying these interactions and responses remain to be explored. Discontinuous sucrose density gradient separation of 1321N1 cells (expressing recombinant hP2Y2R) homogenates revealed co‐fractionation of P2Y2Rs in cav‐1 (+) membrane‐raft fractions. Likewise, confocal microscopy revealed that a significant percent of P2Y2Rs co‐localized with cav‐1 in its sub‐cellular distribution. Blocking cav‐1 expression in hP2Y2‐1321N1 cells elicited abnormal intracellular Ca2+ mobilization responses and uncharacteristic patterns of AKT, ERK1/2 and p38 phosphorylation levels when stimulated with nucleotide agonists, as determined by microfluorometric calcium imaging analyses and immunoblotting, respectively. Our findings suggest that P2Y2Rs reside in membrane caveolae of non‐stimulated 1321N1 cells and that this sub‐cellular compartment may couple its downstream signaling machinery. We hypothesize that P2Y2Rs’ signaling cascades are linked to their expression in cav‐1 (+) micro‐domains. NAM is supported by the NIGMS‐NIH award number R25GM061838 at the UPR‐MSC.

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Glial cells (GC) are the most abundant cellular element in the nervous system. Astrocytes have gained prominence as mediators of acute and persistent inflammatory reactions in the CNS, such as those occuring after spinal cord injury and Alzheimer's disease (AD). GC responses can be both protective and degenerative in nature, depending on their spatiotemporal chemical environment. Thus, understanding reactive gliosis and the molecular mechanisms underlying these responses, and what promotes their glioprotective roles is imperative. Therefore, we have implemented the use of an in vitro Mechanical Strain Injury Model System using 1321N1 human astrocytoma cells. The stable expression of the P2Y2 nucleotide receptor in normally P2‐devoid 1321N1 cells promotes cell survival. Also, data suggests that this receptor interacts with the membrane‐raft protein cav‐1 during signaling/trafficking. Furthermore, the late‐onset AD marker protein sorLA, has also been shown to be a partner of cav‐1 in glia. Therefore, we have hypothesized that the glioprotective actions of both P2Y2 and sorLA are intimately linked to their expression and trafficking via cav‐1 positive membrane rafts micro domains. NAM is supported by the NIGMS‐MBRS‐RISE Program GM61838 at the UPR‐MSC.