The identity of the cell adhesive factors in fetal bovine serum, commonly used to supplement growth media, remains a mystery due to the plethora of serum proteins. In the present analyses, we showed that fetuin-A, whose function in cellular attachment in tissue culture has been debated for many years, is indeed a major serum cell attachment factor particularly for tumor cells. We are able to report this because of a new purification strategy that has for the first time given us a homogeneous protein band in colloidal Coomassie-stained gels that retains biological activity. The tumor cells adhered to immobilized fetuin-A and not ␣ 2 -macroglobulin, its major contaminant. The interaction of cells with fetuin-A was driven mainly by Ca 2؉ ions, and cells growing in regular medium supplemented with fetal bovine serum were just as sensitive to loss of extracellular Ca 2؉ ions as cells growing in fetuin-A. Fractionation of human serum revealed that cell attachment was confined to the fractions that had fetuin-A. Interestingly, the tumor cells also took up fetuin-A and secreted it back to the medium using an unknown mechanism that can be observed in live cells. The attachment of tumor cells to fetuin-A was accompanied by phosphatidylinositol 3-kinase/Akt activation that was down-regulated in cells that lack annexin-A6, one of the cell surface receptors for fetuin-A. Taken together, our data show the significance of fetuin-A in tumor cell growth mechanisms in vitro and open new research vistas for this protein.Serum, particularly fetal bovine serum, is widely used as a supplement in culture media that is required for growth of most cells in culture (for a review, see Ref. 1). In the absence of serum, most cells fail to adhere properly, spread, and grow on culture dishes. Serum has a plethora of adhesion (2-4) and growth factors (5). The prevailing assumption over the past two decades has been that integrins are the major cellular receptors for adhesion in cell cultures (6). Cellular adhesion to extracellular adhesion molecules such as vitronectin, fibronectin, and laminin using integrins requires the divalent ions Mg 2ϩ /Mn 2ϩ (7). In addition, cell signaling cues for spreading and growth mechanisms in anchorage-dependent cells emanate from the interaction of cells with the adhesion molecules (8). Although it is easy to define such signals in situations where cells are allowed to adhere to known purified extracellular matrix proteins such as fibronectin and collagen (9), adhesion in the presence of fetal bovine serum is complex in that any one of the myriad attachment proteins and or growth factors has the potential to mediate adhesion and growth signals.Ever since it was first purified and described, fetuin-A/ ahsg 2 was suspected of being the principal cell adhesion molecule in serum (1, 10). Fetuin-A isolated and purified from serum by the Pedersen method (hereafter referred to as Pedersen fetuin-A) demonstrated cell adhesive properties in the presence of divalent ions (11). The Pedersen fetuin-A, however, is not cons...

Background: DAT regulates dopamine neurotransmission in the brain. Results: ␣-Synuclein influences DA efflux and membrane microdomain distribution of DAT. Conclusion: DAT activation recruits ␣-synuclein to the membrane, which in turn influences dopamine neurotransmission. Significance: Understanding the mechanisms associated with ␣-synuclein regulation of DAT may reveal disease-modifying targets for the treatment of pathologies associated with DA dysregulation.

The duration and strength of the dopaminergic signal is regulated by the dopamine transporter (DAT). Drug addiction, neurodegenerative and neuropsychiatric diseases have all been associated with altered DAT activity. The membrane localization and the activity of DAT are regulated by a number of intracellular proteins. α-synuclein, a protein partner of DAT, is implicated in neurodegenerative disease and drug addiction. Little is known about the regulatory mechanisms of the interaction between DAT and α-synuclein, the cellular location of this interaction, and the functional consequences of this interaction on the basal, amphetamine (AMPH) induced DAT-meditated DA efflux and membrane microdomain distribution of the transporter. Here, we found that the majority of DAT/α-synuclein protein complexes are found at the plasma membrane of dopaminergic neurons or mammalian cells, and that AMPH-mediated increase in DAT activity enhances the association of these proteins at the plasma membrane. Further examination of the interaction of DAT and α-synuclein revealed a transient interaction between these two proteins at the plasma membrane. Additionally, we found DAT-induced membrane depolarization enhances plasma membrane localization of α-synuclein, which in turn increases DA efflux and enhances DAT localization in cholesterol rich membrane microdomains.

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