Little is known concerning the intracellular transport of the G protein-coupled receptors (GPCRs). Previous studies suggested a functional role for those residues immediately preceding the conserved palmitoylated cysteine residues in the intracellular carboxyl termini of some GPCRs in cell surface transport. For the human vasopressin V2 receptor, we assessed the significance of a dileucine sequence with an upstream glutamate residue (ELRSLLCC) in mediating cell surface delivery. A series of deletion and point mutants in this region were constructed, and the mutant receptors were expressed in transiently transfected COS.M6 cells. By using [3H]arginine vasopressin binding assays to intact cells and immunofluorescence studies with intact and permeabilized cells, we show that residues E335 (mutant E335Q) and L339 (mutant L339T) are obligatory for receptor transport to the plasma membrane. Residue L340 has a minor but significant influence. [3H]Arginine vasopressin binding experiments on membranes of lysed cells failed to detect any intracellular binding sites for the transport-deficient mutant receptors, suggesting that residues E335 and L339 participate in receptor folding. Studies with green fluorescent protein-tagged receptors demonstrate that the bulk of the mutant receptors E335Q and L339T are trapped in the endoplasmic reticulum. Complex glycosylation was absent in these mutant receptors, supporting this conclusion. These data demonstrate that the glutamate/dileucine motif of the vasopressin V2 receptor is critical for the escape of the receptor from the endoplasmic reticulum, most presumably by establishing a functional and transport-competent folding state. A databank analysis revealed that these residues are part of a conserved region in the GPCR family.

Some membrane-permeable antagonists restore cell surface expression of misfolded receptors retained in the endoplasmic reticulum (ER) and are therefore termed pharmacochaperones. Whether pharmacochaperones increase protein stability, thereby preventing rapid degradation, or assist folding via direct receptor interactions or interfere with quality control components remains elusive. We now show that the cell surface expression and function (binding of the agonist) of the mainly ER-retained wild-type murine vasopressin V 2 receptor GFP fusion protein (mV 2 R⅐GFP) is restored by the vasopressin receptor antagonists SR49059 and SR121463B with EC 50 values similar to their K D values. This effect was preserved when protein synthesis was abolished. In addition, SR121463B rescued eight mutant human V 2 Rs (hV 2 Rs, three are responsible for nephrogenic diabetes insipidus) characterized by amino acid exchanges at the C-terminal end of transmembrane helix TM I and TM VII. In contrast, mutants with amino acid exchanges at the interface of TM II and IV were not rescued by either antagonist. The mechanisms involved in successful rescue of cell surface delivery are explained in a three-dimensional homology model of the antagonist-bound hV 2 R.Water homeostasis in mammals is regulated through arginine-vasopressin (AVP), 1 acting through the vasopressin V 2 receptor (V 2 R) expressed in the renal collecting duct (1). In Xlinked nephrogenic diabetes insipidus (NDI), the kidney shows a resistance to the action of AVP, caused by inactivating mutations of the human V 2 R (hV 2 R) gene (2). More than 150 different mutations have been described (for review, see Ref.3), 50% of which are missense mutations resulting in the substitution of a single amino acid. Most of the hV 2 R mutants with a single amino acid exchange are retained within the ER and not transported to the cell surface (for review, see Ref.3). Most likely, the amino acid exchanges result in improper folding of the mutant hV 2 Rs and subsequently prolonged association with molecular chaperones. For example, for the NDI mutant hR337X, a prolonged association with the ER-chaperone calnexin has been observed (4). Chaperone association prevents the aggregation of misfolded proteins, but also inhibits the exit of improperly folded proteins from the ER until correct folding is established.Recently, it has been found that membrane-permeable antagonists not only inhibit receptor activation, but also promote cell surface expression of misfolded, ER-retained G proteincoupled receptors (GPCRs). This concept represents an intriguing new approach for the therapy of congenital diseases caused by mutations in genes encoding GPCRs. For the ER-retained rhodopsin mutant P23H (a frequent cause of autosomal-dominant retinitis pigmentosa), it has been shown in vitro that the inverse agonist 9-cis-retinal or the non-hydrolyzable inverse agonist 11-cis-7-ring-retinal promoted cell surface expression (5,6). Restoration of cell surface expression by antagonists or inverse agonists has also been...

Tumor immunology has received a large impetus from the identification of tumor-associated antigens. Among them, a monoclonal antibody, 22.1.1, was instrumental in defining a novel tumor-associated antigen that was termed "receptor binding cancer antigen expressed on SiSo cells" (RCAS1). RCAS1 was proposed to induce growth arrest and apoptosis on activated immune cells, mediated by a putative death receptor. Structurally, RCAS1 was predicted to exist as a type II transmembrane protein and in a soluble form. Here, we analyzed occurrence, membrane topology, and subcellular localization of the RCAS1-encoded gene product. RCAS1 was shown to be a ubiquitously expressed type III transmembrane protein with a Golgi-predominant localization. Monoclonal antibody 22.1.1 failed to recognize RCAS1, as demonstrated by confocal microscopy. Instead, we showed that the cognate 22.1.1 epitope is identical with the tumor-associated O-linked glycan Tn (N-acetyl-D-galactosamine, GalNAc). Overexpression of RCAS1 in cell lines that are negative for 22.1.1 surface staining led to the generation of Tn and the closely related TF (Thomsen-Friedenreich, Gal␤1-3GalNAc) antigen, thus providing a functional link to the generation of the 22.1.1 epitope. We suggest that RCAS1 modulates surface expression of tumor-associated, normally cryptic O-linked glycan structures and contributes indirectly to the antigenicity of tumor cells.

Objective To characterize the natural history of spinal muscular atrophy (SMA) over 24 months using innovative measures such as wearable devices, and to provide evidence for the sensitivity of these measures to determine their suitability as endpoints in clinical trials. Methods Patients with Type 2 and 3 SMA (N = 81) with varied functional abilities (sitters, nonsitters, nonambulant, and ambulant) who were not receiving disease‐modifying treatment were assessed over 24 months: motor function (Motor Function Measure [MFM]), upper limb strength (MyoGrip, MyoPinch), upper limb activity (ActiMyo®), quantitative magnetic resonance imaging (fat fraction [FFT2] mapping and contractile cross‐sectional area [C‐CSA]), pulmonary function (forced vital capacity [FVC], peak cough flow, maximum expiratory pressure, maximum inspiratory pressure, and sniff nasal inspiratory pressure), and survival of motor neuron (SMN) protein levels. Results MFM32 scores declined significantly over 24 months, but not 12 months. Changes in upper limb activity could be detected over 6 months and continued to decrease significantly over 12 months, but not 24 months. Upper limb strength decreased significantly over 12 and 24 months. FVC declined significantly over 12 months, but not 24 months. FFT2 increased over 12 and 24 months, although not with statistical significance. A significant increase in C‐CSA was observed at 12 but not 24 months. Blood SMN protein levels were stable over 12 and 24 months. Interpretation These data demonstrate that the MFM32, MyoGrip, MyoPinch, and ActiMyo® enable the detection of a significant decline in patients with Type 2 and 3 SMA over 12 or 24 months.