Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are structurally related lipid mediators that act on distinct Gprotein-coupled receptors to evoke similar responses, including Ca# + mobilization, adenylate cyclase inhibition, and mitogenactivated protein (MAP) kinase activation. However, little is still known about the respective receptors. A recently cloned putative LPA receptor (Vzg-1\Edg-2) is similar to an orphan G icoupled receptor termed Edg-1. Here we show that expression of
We have isolated a mouse cDNA of 5.4 kb, encoding a new membee of the family of receptor-like protein tyrosine phospkatases, termed mRPTPy. The cDNA predicts a protein of 1432 amino acids (not including signal peptide) with a calculated M, of 161 636. In addition, we have cloned the human homologue, hRPTPp, which shows 98.7% amino acid identity to mRPTP& The predicted mRPTP# protein consists of a 722 amino acid cxtracellular region, containing 13 potential N-glycosylation sites, a single transmembrane domain and a G88 amino acid intracellular part containing 2 tandem repeats homologous to the catalytic domains of other tyrosine phosphatases. The N-terminal extracellular pact contains a region of about 170 amino acids with no sequence similarities to known proteins, followed by one Q-like domain and 4 fibronectin type III-like domains. The intracellular part is unique in that the region between the transmambrane domain and the first catalytic dormain is aboui iii;iz ss large as in other receptor-like protein tyrosine phosphatases. RNA blot analysis reveals a single transcript, that is most abundant in lung and present in much lower amounts in brain and heart. Transfection of the mRPTPp cDNA into COS cells results in the synthesis of a protein with an apparent M, of 195 000, as detected in immunoblots using an antipeptide antibody. The human RPTPp gene is localized on chromosome Ilpter-ql 1, a region with frequent abnormalities implicated in human cancer.Receptor-like tyrosine phosphatase I. INTROIX_JCTION Reversible protein phosphorylation has long been considered a key mechanism for regulating cellular processes [l]. While the majority of protein phosphorylation occurs at serine and threonine residues, phosphorylation at tyrosine residues is attracting a great deal of interest since the discovery that many oncogene products and growth factor receptors posses intrinsic protein tyrosine kinase (PTK) activity. The importance of tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transforma.tion is now well established [Z-5]. Tyrosine phosphorylated proteins can be specifically dephosphorylated through the action of protein tyrosine phosphatases (PTPs) cific RPTP, CD45, provide direct support for that view t271.
The G protein-linked receptor for neurokinin A (NKA) couples to stimulation of phospholipase C and, in some cells, adenylyl cyclase. We have examined the function of the C-terminal cytoplasmic domain in receptor signaling and desensitization. We constructed C-terminal deletion mutants of the human NK-2 receptor (epitope tagged) to remove potential Ser/Thr phosphorylation sites, and expressed them in both mammalian and insect cells. When activated, truncated receptors mediate stronger and more prolonged phosphoinositide hydrolysis than wild-type receptor; however, the amplitude and kinetics of the NKA-induced rise in cytosolic Ca2+ remain unaltered. Protein kinase C (PKC)-activating phorbol ester abolishes wild-type receptor signaling but not mutant receptor signaling. Mutant receptors also mediate enhanced and prolonged cAMP generation, at least in part via PKC activation. When expressed in COS cells or Sf9 insect cells, the wild-type receptor is phosphorylated; receptor phosphorylation increases after addition of either NKA or phorbol ester. In contrast, mutant receptors are not phosphorylated by either treatment. Our results suggest that C-terminal Ser/Thr phosphorylation sites in the NK-2 receptor have a critical role in both homologous and heterologous desensitization. Removal of these phosphorylation sites results in a receptor that mediates sustained activation of signaling pathways and is insensitive to inhibition by PKC.
RPTP is a receptor-like protein-tyrosine phosphatase (RPTP) whose ectodomain mediates homotypic cellcell interactions. The intracellular part of RPTP contains a relatively long juxtamembrane domain (158 amino acids; aa) and two conserved phosphatase domains (C1 and C2). The membrane-proximal C1 domain is responsible for the catalytic activity of RPTP, whereas the membrane-distal C2 domain serves an unknown function. The regulation of RPTP activity remains poorly understood, although dimerization has been proposed as a general mechanism of inactivation. Using the yeast two-hybrid system, we find that the C1 domain binds to an N-terminal noncatalytic region in RPTP, termed JM (aa 803-955), consisting of a large part of the juxtamembrane domain (120 aa) and a small part of the C1 domain (33 aa). When co-expressed in COS cells, the JM polypeptide binds to both the C1 and the C2 domain. Strikingly, the isolated JM polypeptide fails to interact with either full-length RPTP or with truncated versions of RPTP that contain the JM region, consistent with the JM-C1 and JM-C2 interactions being intramolecular rather than intermolecular. Furthermore, we find that large part of the juxtamembrane domain (aa 814 -922) is essential for C1 to be catalytically active. Our findings suggest a model in which RPTP activity is regulated by the juxtamembrane domain undergoing intramolecular interactions with both the C1 and C2 domain. Protein-tyrosine phosphatases (PTPs)1 play important roles in signal transduction pathways regulated by tyrosine phosphorylation. Members of the superfamily of PTPs use the same catalytic mechanism and are broadly classified into transmembrane or receptor-like PTPs (RPTPs) and intracellular, nonreceptor PTPs (reviewed in Refs. 1 and 2). Members of the RPTP subfamily are type I membrane proteins consisting of a variable ectodomain, a single membrane-spanning region, and in most cases, two conserved intracellular phosphatase domains. The RPTPs are further classified according to the structure of their ectodomains (reviewed in Refs. 3 and 4). The large variety in ectodomain structure suggests the existence of an equal number of putative ligands, yet in most cases the corresponding ligands have not been identified.RPTP is the prototype member of a subfamily of RPTPs that mediate homophilic cell-cell interactions via their ectodomains and, hence, are thought to play a role in cell adhesionmediated processes (5-8). The ectodomain of RPTP shows similarities with that of cell-cell adhesion molecules and consists of an N-terminal "MAM" domain, which is critical for mediating cell-cell adhesion (9), followed by an Ig-like domain and four fibronectin type III repeats (10). Its intracellular part consists of a juxtamembrane domain of 158 amino acids (aa), which is relatively long compared with that in other RPTPs, and two tandem phosphatase domains referred to as C1 and C2. As in most other RPTPs, the membrane-proximal C1 domain of RPTP is catalytically active, whereas the membranedistal C2 domain shows no activity, a...
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