Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin

Abstract

Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells.For more than 30 years, heparin has been known to specifically bind to cells in the vasculature and alter their physiology in addition to its well recognized function as an anticoagulant. Heparin binds to many proteins, including numerous growth factors, cytokines, coagulation factors, cell adhesion molecules, growth factor receptors, matrix glycoproteins, and others (for a review, see Ref. 1). In fact, heparin and the closely related glycosaminoglycan heparan sulfate (HS), 4 interact with more than 400 proteins (2). Heparin decreases endothelial cell (EC) inflammatory gene expression and slows vascular smooth muscle cell (VSMC) proliferation (reviewed in Ref. 3). Specifically, ECs bind and endocytose heparin (4, 5), which is followed by decreased inflammatory signaling through NF-B (6) and stress kinase activity (7,8). Heparin binding in VSMCs (9) results in decreases in growth factor-induced ERK signaling (10, 11), inhibition of downstream transcription factor activity (12-14), changes in cell cycle inhibitory factors (15), and decreased proliferation (10, 16).Reports of fluorescent heparin uptake into cells, where it modulated transcription factor function (17), and the requirements of HSPGs for basic growth factor delivery to the nucleus (18) indicate that receptor-mediated uptake of heparin or HS may also be critical for some heparin effects. Similarly, shed HSPG syndecan-1 can be taken up by cells and transported to the nucleus, where it alters histone acetylation (19). HS chains are required for uptake, and this uptake can be inhibited by exogenously added heparin. It is likely that the uptake of highly charged heparin and HS chains involves a receptor to manage transport across the membrane. Although many heparin-interacting proteins have been linked to specific functions, a receptor respon...

Heparin Decreases in Tumor Necrosis Factor α (TNFα)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1

“…The observations that pretreatment with antibodies to a putative heparin receptor decreases TNF␣-induced JNK and p38 activation and decreases stress fiber formation are consistent with previous reports showing that these antibodies mimic the effects of heparin in VSMCs (19,22,24). Here we provide functional evidence that the anti-inflammatory effects of heparin in ECs are mediated through the heparin receptor TMEM184A, as seen in VSMCs (28). TMEM184A knockdown interferes with heparin responses, confirming our HRmAb evidence.…”

“…VSMCs eliminated heparin responses (28). To determine whether this heparin-responsive protein identified with our HRmAbs and confirmed in VSMC is also responsible for the heparin effects in ECs reported here, we first confirmed that TMEM184A is present in RAOECs across the range of cell density we studied (Fig.…”

“…The accompanying report identifies TMEM184A as the heparin-interacting protein to which the HRmAbs bind (28). Knockdown of TMEM184A in VSMCs eliminates heparin responses (28). Here we report evidence that unfractionated heparin treatment of ECs results in decreased JNK and p38 activity and that HRmAbs mimic heparin effects on JNK and p38 activity.…”

“…Knockdown of TMEM184A Interferes with Heparin Responses-We have recently identified TMEM184A to be a heparin-responsive transmembrane protein required for heparin effects in VSMCs (28). The identification of TMEM184A also involved the use of HRmAbs that mimic effects of heparin in ECs (Figs.…”

“…The accompanying report identifies TMEM184A as the heparin-interacting protein to which the HRmAbs bind (28). Knockdown of TMEM184A in VSMCs eliminates heparin responses (28).…”