Matriptase-2 is a member of the TTSPs (type II transmembrane serine proteases), an emerging class of cell surface proteases involved in tissue homoeostasis and several human disorders. Matriptase-2 exhibits a domain organization similar to other TTSPs, with a cytoplasmic N-terminus, a transmembrane domain and an extracellular C-terminus containing the non-catalytic stem region and the protease domain. To gain further insight into the biochemical functions of matriptase-2, we characterized the subcellular localization of the monomeric and multimeric form and identified cell surface shedding as a defining point in its proteolytic processing. Using HEK (human embryonic kidney)-293 cells, stably transfected with cDNA encoding human matriptase-2, we demonstrate a cell membrane localization for the inactive single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric, as well as multimeric, forms covalently linked by disulfide bonds. Furthermore, matriptase-2 undergoes shedding into the conditioned medium as an activated two-chain form containing the catalytic domain, which is cleaved at the canonical activation motif, but is linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by MS, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active-site serine residue is dependent on the catalytic activity of co-expressed or co-incubated wild-type matriptase-2, indicating a transactivation and trans-shedding mechanism.

Endothelin‐1 (ET‐1) plays an important role in tissue remodelling and fibrogenesis by inducing synthesis of collagen I via protein kinase C (PKC). ET‐1 signals are transduced by two receptor subtypes, the ETA‐ and ETB‐receptors which activate different Gα proteins. Here, we investigated the expression of both ET‐receptor subtypes in human primary dermal fibroblasts and demonstrated that the ETA‐receptor is the major ET‐receptor subtype expressed. To determine further signalling intermediates, we inhibited Gαi and three phospholipases. Pharmacologic inhibition of Gαi, phosphatidylcholine‐phospholipase C (PC‐PLC) and phospholipase D (PLD), but not of phospholipase Cβ, abolished the increase in collagen I by ET‐1. Inhibition of all phospholipases revealed similar effects on TGF‐β1 induced collagen I synthesis, demonstrating involvement of PC‐PLC and PLD in the signalling pathways elicited by ET‐1 and TGF‐β1. ET‐1 and TGF‐β1 each stimulated collagen I production and in an additive manner. ET‐1 further induced connective tissue growth factor (CTGF), as did TGF‐β1, however, to lower levels. While rapid and sustained CTGF induction was seen following TGF‐β1 treatment, ET‐1 increased CTGF in a biphasic manner with lower induction at 3 h and a delayed and higher induction after 5 days of permanent ET‐1 treatment. Coincidentally at 5 days of permanent ET‐1 stimulation, a switch in ET‐receptor subtype expression to the ETB‐receptor was observed. We conclude that the signalling pathways induced by ET‐1 and TGF‐β1 leading to augmented collagen I production by fibroblasts converge on a similar signalling pathway. Thereby, long‐time stimulation by ET‐1 resulted in a changed ET‐receptor subtype ratio and in a biphasic CTGF induction.

Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ET A and ET B . To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential Ga protein binding capacity. Endothelin-1 stimulation of wild-type ET A or ET B induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas fulllength nonpalmitoylated ET A and ET B mutants failed to stimulate ERK. A truncated ET B lacking the C-terminal tail domain including putative phosphorylation and arrestin binding site(s) but retaining the critical palmitoylation site(s) was still able to fully stimulate ERK activation. Using mutated ET receptors with selective G-protein-coupling we found that endothelin-induced stimulation of Ga q , but not of

Post-translational modifications such as phosphorylation and palmitoylation play important roles for the function and regulation of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins. Here we demonstrate that the human endothelin receptor A (ET A ) incorporates [ 3 H]palmitate. Mutation of a cluster of five cysteine residues present in the cytoplasmic tail of ET A into serine or alanine residues completely prevented palmitoylation of the receptor. The ligand binding affinity of the non-palmitoylated ET A mutants was essentially unchanged as compared to the palmitoylated wild type ET A suggesting that the replacement of the cysteine residues did not alter the overall structure of the receptor. Furthermore, the ligand-induced stimulation of adenylyl cyclase by the mutant ET A was unaffected by the mutation. In contrast, the mutated nonpalmitoylated receptors but not the wild type receptor failed to stimulate phosphatidylinositol hydrolysis by phospholipase C activation upon challenge by endothelin-1. Furthermore, the mutant receptors failed to stimulate the ligand-induced transient increase in the cytoplasmic calcium seen with the wild type ET A . Endothelin-1 induced mitogenic stimuli via the wild type receptors but not through the mutated receptors suggesting an important role for phospholipase C in this signal transduction pathway. The differential regulation of distinct signal transduction pathways by posttranslational modification suggests that palmitoylation of the ET A provides a novel mechanism of modulating ET A receptor activity.Post-translational modifications of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins (G protein) modulate receptor function and activity (1-3). One such modification is the palmitoylation of conserved cysteine residues located in the cytoplasmic tail of many G protein-coupled receptors. It has been suggested that this acylation provides a membrane anchor that creates a fourth cytoplasmic receptor loop (4, 5). The functional role of palmitoylation has been studied for adrenergic receptors in some detail. Prevention of palmitoylation of a conserved cysteine residue, Cys 341 , of the ␤-adrenergic receptor causes functional uncoupling of the receptor from the adenylyl cyclase pathway (4). Furthermore, the nonpalmitoylated receptor shows an increased basal phosphorylation and a rapid desensitization in response to its ligand (6). Activation of the ␤ 2 -adrenergic receptor results in an enhanced palmitoylation thereby increasing the amount of functionally coupled receptor (7). Adrenergic receptor activation also increases palmitoylation of a receptor-associated stimulatory G protein (8). These findings clearly show the dynamic properties of protein/receptor palmitoylation. Unlike the ␤ 2 -adrenergic receptor a mutation of the corresponding palmitoylation site in the ␣ 2A -adrenergic receptor did not influence the signal transduction activity but decreased the ligand-promoted down-regulation of the receptor (9). On the other hand, purified nonpa...

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