The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2A Prevents Receptor Oligomerization

N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF(2(a))R and the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF(2(a))R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.     

The signal peptide of the G protein-coupled human endothelin B receptor is necessary for translocation of the N-terminal tail across the endoplasmic reticulum membrane

The initial step of the intracellular transport of G protein-coupled receptors, their insertion into the membrane of the endoplasmic reticulum, follows one of two different pathways. Whereas one group uses the first transmembrane domain of the mature receptor as an uncleaved signal anchor sequence for this process, a second group possesses additional cleavable signal peptides. The reason this second subset requires the additional signal peptide is not known. Here we have assessed the functional significance of the signal peptide of the endothelin B (ET(B)) receptor in transiently transfected COS.M6 cells. A green fluorescent protein-tagged ET(B) receptor mutant lacking the signal peptide was nonfunctional and retained in the endoplasmic reticulum, suggesting that it has a folding defect. To determine the defect in more detail, ET(B) receptor fragments containing the N-terminal tail, first transmembrane domain, and first cytoplasmic loop were constructed. We assessed N tail translocation across the endoplasmic reticulum membrane in the presence and absence of a signal peptide and show that the signal peptide is necessary for N tail translocation. We postulate that signal peptides are necessary for those G protein-coupled receptors for which post-translational translocation of the N terminus is impaired or blocked by the presence of stably folded domains.     

The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2a Decreases Receptor Expression and Prevents Gi-mediated Inhibition of Adenylyl Cyclase Activity

The corticotropin-releasing factor receptor type 2a (CRF_2(a)R) belongs to the family of G protein-coupled receptors. The receptor possesses an N-terminal pseudo signal peptide that is unable to mediate targeting of the nascent chain to the endoplasmic reticulum membrane during early receptor biogenesis. The pseudo signal peptide remains uncleaved and consequently forms an additional hydrophobic receptor domain with unknown function that is unique within the large G protein-coupled receptor protein family. Here, we have analyzed the functional significance of this domain in comparison with the conventional signal peptide of the homologous corticotropin-releasing factor receptor type 1 (CRF₁R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF_2(a)R in comparison with the CRF₁R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the G_s protein, G_i-mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF₁R in signal peptide exchange experiments. Taken together, our results show that signal peptides do not only influence early protein biogenesis. In the case of the corticotropin-releasing factor receptor subtypes, the use of conventional and pseudo signal peptides have an unexpected influence on signal transduction.     

Presence of a N-terminal signal peptide in class II G protein-coupled receptors: crucial role for expression of the human VPAC1 receptor

The hVPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase activating peptide (PACAP) has an N-terminal signal peptide like all other class II G protein-coupled receptors (GPCRs). We determined the role of the signal peptide in expression of human VPAC1 receptor in transfected CHO cells. Three constructs were transfected: Flag30-hVPAC1, a receptor containing an inserted FLAG sequence between Ala30 and Ala31 and fused in the C-terminal position to GFP; Flag30-[delta1-30]-hVPAC1, the same construct as Flag30-hVPAC1 but lacking the 1-30 putative signal peptide (SP) sequence; Flag0-hVPAC1, a receptor containing an N-terminal FLAG sequence and fused in the C-terminal position to GFP. For each construct, we determined 125I-VIP binding, VIP-induced cAMP production, GFP fluorescence and indirect immunofluorescence on nonpermeabilized cells incubated with mouse monoclonal anti-Flag antibodies. The data were consistent with a crucial role of the signal peptide for expression of functional VPAC1 receptors at the cell surface and suggested that the signal peptide is cleaved during the translocation of the receptor to the plasma membrane, probably in the endoplasmic reticulum.     

Membrane protein topology of oleosin is constrained by its long hydrophobic domain.

Oleosin proteins from Arabidopsis assume a unique endoplasmic reticulum (ER) topology with a membrane-integrated hydrophobic (H) domain of 72 residues, flanked by two cytosolic hydrophilic domains. We have investigated the targeting and topological determinants present within the oleosin polypeptide sequence using ER-derived canine pancreatic microsomes. Our data indicate that oleosins are integrated into membranes by a cotranslational, translocon-mediated pathway. This is supported by the identification of two independent functional signal sequences in the H domain, and by demonstrating the involvement of the SRP receptor in membrane targeting. Oleosin topology was manipulated by the addition of an N-terminal cleavable signal sequence, resulting in translocation of the N terminus to the microsomal lumen. Surprisingly, the C terminus failed to translocate. Inhibition of C-terminal translocation was not dependent on either the sequence of hydrophobic segments in the H domain, the central proline knot motif or charges flanking the H domain. Therefore, the topological constraint results from the length and/or the hydrophobicity of the H domain, implying a general case that long hydrophobic spans are unable to translocate their C terminus to the ER lumen.     

The hydrophobic amino acid residues in the membrane-proximal C tail of the G protein-coupled vasopressin V2 receptor are necessary for transport-competent receptor folding

It is believed that the membrane-proximal C tail of the G protein-coupled receptors forms an additional alpha helix with amphipathic properties (helix 8). It was previously shown for the vasopressin V2 receptor (V2R) that a conserved dileucine motif (L(339), L(340)) in this putative helix 8 is necessary for endoplasmic reticulum (ER) to Golgi transfer of the receptor. Here, we demonstrate that the other hydrophobic residues forming the non-polar side of this helix (F(328), V(332) and L(336)) are also transport-relevant. In contrast, the multiple serine residues contributing to the more hydrophilic side (S(330), S(331), S(333), S(334), S(338)) do not influence receptor trafficking. In addition, we show unambiguously by the use of pharmacological chaperones that the hydrophobic residues of the putative helix 8 do not form a transport signal necessary for receptor sorting into ER to Golgi vesicles. Instead, they are necessary to establish a transport-competent folding state in the early secretory pathway.     

Identification of the endoplasmic reticulum targeting signal in vesicle-associated membrane proteins

The vesicle-associated membrane proteins (Vamp(s)) function as soluble N-ethylmaleimide-sensitive factor attachment receptor proteins in the intracellular trafficking of vesicles. The membrane attachment of Vamps requires a carboxyl-terminal hydrophobic sequence termed an insertion sequence. Unlike other insertion sequence-containing proteins, targeting of the highly homologous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP and a membrane-bound receptor. To determine if this mechanism of targeting to the endoplasmic reticulum extends to other Vamps, we compared the membrane binding of Vamp1 and Vamp2 with the distantly related Vamp8. Similar to the other Vamps, Vamp8 requires both ATP and a membrane component to target to the endoplasmic reticulum. Furthermore, binding curves for the three Vamps overlap, suggesting a common receptor-mediated process. We identified a minimal endoplasmic reticulum targeting domain that is both necessary and sufficient to confer receptor-mediated, ATP-dependent, binding of a heterologous protein to microsomes. Surprisingly, this conserved sequence includes four positively charged amino acids spaced along an amphipathic sequence, which unlike the carboxyl-terminal targeting sequence in mitochondrial Vamp isoforms, is amino-terminal to the insertion sequence. Because Vamps do not bind to phospholipid vesicles, it is likely that these residues mediate an interaction with a protein, rather than bind to acidic phospholipids. Therefore, we suggest that a bipartite motif is required for the specific targeting and integration of Vamps into the endoplasmic reticulum with receptor-mediated recognition of specifically configured positive residues leading to the insertion of the hydrophobic tail into the membrane.     

Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets

Although caveolins normally reside in caveolae, they can accumulate on the surface of cytoplasmic lipid droplets (LDs). Here, we first provided support for our model that overaccumulation of caveolins in the endoplasmic reticulum (ER) diverts the proteins to nascent LDs budding from the ER. Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs. We used the fact that wild-type caveolin-1 accumulates in LDs after brefeldin A treatment or when linked to an ER retrieval motif to search for mutants defective in LD targeting. The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1. Certain Leu insertions blocked LD targeting, independently of hydrophobic domain length, but dependent on their position in the domain. We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1.     

Rer1, a putative transmembrane domain receptor responsible for targeting proteins to the endoplasmic reticulum

Summary Localization of resident proteins provides identity to subcellular compartments. Most proteins depend on a combination of both retention and retrieval to maintain their steady-state distribution. Rerl is a putative receptor protein mediating retrieval of membrane proteins of the endoplasmic reticulum. This retrieval relies on an unusual hydrophobic target sequence, the transmembrane domain. Apart from Rerl, coatomer is also required to retrieve escaped membrane proteins from the early Golgi region back to the endoplasmic reticulum. Current evidence suggests that the Rerl-mediated retrieval of membrane proteins is a general sorting pathway in eukaryotic cells contributing to the maintenance of compartmental identity in the early secretory pathway.     

Trafficking of lysosomal enzymes

The targeting of lysosomal enzymes from their site of synthesis in the rough endoplasmic reticulum (RER) to their final destination in lysosomes is directed by a series of protein and carbohydrate recognition signals on the enzymes. Lysosomal enzymes, along with secretory and plasma membrane proteins, contain amino-terminal signal sequences that direct the vectorial discharge of the nascent proteins into the lumen of the RER. The three classes of proteins also share a common peptide signal for asparagine glycosylation. The next signal is unique to lysosomal enzymes and permits their high-affinity binding to a specific phosphotransferase that catalyzes the formation of the mannose 6-phosphate recognition marker. This carbohydrate determinant allows binding to specific receptors that translocate the lysosomal enzymes from the Golgi complex to an acidified prelysosomal compartment. There the lysosomal enzymes are discharged for final packaging into lysosomes. Two distinct mannose 6-phosphate receptors have been identified, and cDNAs encoding their entire sequences have been cloned. An analysis of the deduced amino acid sequences of the receptors shows that each is composed of four structural domains: a signal sequence, an extracytoplasmic amino-terminal domain, a hydrophobic membrane-spanning region, and a cytoplasmic domain. The entire extracytoplasmic region of the small receptor is homologous to the 15 repeating domains that constitute the extracytoplasmic portion of the large receptor.     

An Artificial Mitochondrial Tail Signal/Anchor Sequence Confirms a Requirement for Moderate Hydrophobicity for Targeting

Tail-anchored proteins are a group of membrane proteins oriented with their amino terminus in the cytoplasm and their carboxy terminus embedded in intracellular membranes. This group includes the apoptosis-mediating proteins of the Bcl-2 family as well as the vesicle targeting proteins of the SNARE group, among others. A stretch of hydrophobic amino acids at the extreme carboxy terminus of these proteins serves both as a membrane anchor and as a targeting signal. Tail-anchored proteins are differentially targeted to either the endoplasmic reticulum or the mitochondrial outer membrane and the mechanism which accomplishes this selective targeting is poorly understood. Here we define important characteristics of the signal/anchor region which directs proteins to the mitochondrial outer membrane. We have created an artificial sequence consisting of a stretch of 16 leucines bounded by positively charged amino acids. Using this template we demonstrate that moderate hydrophobicity distinguishes the mitochondrial tail-anchor sequence from that of the endoplasmic reticulum tail-anchor sequence. A change as small as introduction of a single polar residue into a sequence that otherwise targets to the endoplasmic reticulum can substantially switch targeting to the mitochondrial outer membrane. Further we show that a mitochondrially targeted tail-anchor has a higher propensity for the formation of alpha-helical structure than a sequence directing tail-anchored proteins to the endoplasmic reticulum.     

A functional interaction between the signal peptide and the translation apparatus is detected by the use of a single point mutation which blocks translocation across mammalian endoplasmic reticulum

A functional interaction between the signal sequence and the translation apparatus which may serve as a first step in chain targeting to the membrane is described. To this end, we exploited the powerful technique of molecular cloning in a procaryotic system and the well characterized translocation system of mammalian endoplasmic reticulum. The signal peptide of subunit B of the heat labile enterotoxin of Escherichia coli (EltB) was fused to several proteins. Single base substitutions were introduced in the signal peptide and their effect on protein synthesis and translocation was studied. We sought a single amino acid substitution which may define certain steps in the coordinated regulation of chain synthesis and targeting to the membrane. The substitution of proline for leucine at residue -8 in the signal peptide abolished all known functions of the signal peptide. In contrast to wild type signal peptide, the mutant signal peptide did not lead to arrest of nascent chain synthesis by signal recognition particle or translocation of the precursor protein across the membrane of the endoplasmic reticulum. Furthermore, the mutant signal peptide was not cleaved by purified E. coli signal peptidase. Interestingly, the mutation resulted in about a 2-fold increase in the rate of synthesis of the precursor protein, suggesting a role for the signal peptide in regulating the synthesis of the nascent secretory chain as a means of ensuring early and efficient targeting of this chain to the membrane. This role might involve interaction of the signal peptide with components of the translation apparatus and/or endogenous signal recognition particle. These results were obtained with three different fusion proteins carrying the signal peptide of EltB thus leading to the conclusion that the effect of the mutation on the structure and function of the signal peptide is independent of the succeeding sequence to which the signal peptide is attached.     

Cloning and sequencing of human FSH receptor cDNA.

We have isolated and sequenced a cDNA encoding the follicle stimulating hormone (FSH) receptor. The deduced amino acid sequence (678 residues) containing seven putative transmembrane segments which displays sequence similarity to G protein-coupled receptors. The receptor consists of 359 residue extracellular domain which contains four N-linked glycosylation sites. While the protein is 89% identical overall with the previously cloned rat FSH receptor, the most highly conserved regions are the putative transmembrane segments (95% similarity).     

The endoplasmic reticulum (ER) translocon can differentiate between hydrophobic sequences allowing signals for glycosylphosphatidylinositol anchor addition to be fully translocated into the ER lumen

The signal sequence within polypeptide chains that designates whether a protein is to be anchored to the membrane by a glycosylphosphatidylinositol (GPI) anchor is characterized by a carboxyl-terminal hydrophobic domain preceded by a short hydrophilic spacer linked to the GPI anchor attachment (omega) site. The hydrophobic domain within the GPI anchor signal sequence is very similar to a transmembrane domain within a stop transfer sequence. To investigate whether the GPI anchor signal sequence is translocated across or integrated into the endoplasmic reticulum membrane we studied the translocation, GPI anchor addition, and glycosylation of different variants of a model GPI-anchored protein. Our results unequivocally demonstrated that the hydrophobic domain within a GPI signal cannot act as a transmembrane domain and is fully translocated even when followed by an authentic charged cytosolic tail sequence. However, a single amino acid change within the hydrophobic domain of the GPI-signal converts it into a transmembrane domain that is fully integrated into the endoplasmic reticulum membrane. These results demonstrated that the translocation machinery can recognize and differentiate subtle changes in hydrophobic sequence allowing either full translocation or membrane integration.     

Post-translational import of the prion protein into the endoplasmic reticulum interferes with cell viability: a critical role for the putative transmembrane domain

Aberrant folding of the mammalian prion protein (PrP) is linked to prion diseases in humans and animals. We show that during post-translational targeting of PrP to the endoplasmic reticulum (ER) the putative transmembrane domain induces misfolding of PrP in the cytosol and interferes with its import into the ER. Unglycosylated and misfolded PrP with an uncleaved N-terminal signal sequence associates with ER membranes, and, moreover, decreases cell viability. PrP expressed in the cytosol, lacking the N-terminal ER targeting sequence, also adopts a misfolded conformation; however, this has no adverse effect on cell growth. PrP processing, productive ER import, and cellular viability can be restored either by deleting the putative transmembrane domain or by using a N-terminal signal sequence specific for co-translational ER import. Our study reveals that the putative transmembrane domain features in the formation of misfolded PrP conformers and indicates that post-translational targeting of PrP to the ER can decrease cell viability.     

The transmembrane domain of receptor-activity-modifying protein 1 is essential for the functional expression of a calcitonin gene-related peptide receptor

Three receptor-activity-modifying proteins (RAMP) define specific interactions between calcitonin (CT) gene-related peptide (CGRP), adrenomedullin (AM) and amylin, and a CT receptor or a CT receptor-like receptor (CRLR). Both form heterodimeric RAMP/receptor complexes at the cell surface. This association represents a novel principle of G protein-coupled receptor function. RAMP1 is transported to the cell surface together with the CRLR or the CT receptor. Here, we have investigated the functional relevance of the short C-terminal intracellular tail QSKRTEGIV and of the single transmembrane domain of human (h) RAMP1 for their interactions with the hCRLR to constitute a CGRP receptor. To this end, hRAMP1 has been sequentially truncated from the C-terminus, and [(125)I]h alpha CGRP/hRAMP1/hCRLR association at the cell surface and cAMP accumulation in response to h alpha CGRP have been examined. With the C-terminal truncation of hRAMP1 by four amino acids wild-type hRAMP1 function was maintained, and the hCRLR was required for the transport of hRAMP1 to the cell surface. Further truncation of hRAMP1 through removal of the remaining five intracellular amino acids revealed CRLR-independent cell surface delivery but otherwise normal hRAMP1 activity. Sequential shortening of the hRAMP1 transmembrane domain resulted in progressively impaired association with the hCRLR and, as a consequence, abolished CGRP receptor function. In conclusion, the intracellular QSKRT sequence adjacent to the transmembrane domain of hRAMP1 provides a signal for intracellular retention. The sequence is unrelated to consensus endoplasmic reticulum retention/retrieval motives and overridden by the presence of the hCRLR. The entire single transmembrane domain of hRAMP1 together with one hydrophilic amino acid residue at its C-terminus is required for the formation of a fully functional CGRP/hRAMP1/hCRLR receptor complex.     

The signal recognition particle receptor alpha subunit assembles co-translationally on the endoplasmic reticulum membrane during an mRNA-encoded translation pause in vitro.

Many proteins, including the alpha subunit of the signal recognition particle receptor (SR alpha), are targeted within the cell by poorly defined mechanisms. A 140 residue N-terminal domain of SR alpha targets and anchors the polypeptide to the endoplasmic reticulum membrane by a mechanism independent of the pathway involving the signal recognition particle. To investigate the mechanism of membrane anchoring, translation pause sites on the SR alpha mRNA were used to examine the targeting of translation intermediates. A strong pause site at nucleotide 507 of the mRNA open reading frame corresponded with the shortest nascent SR alpha polypeptide able to assemble on membranes. An mRNA sequence at this pause site that resembles a class of viral -1 frameshift sequences caused translation pausing when transferred into another mRNA context. Site-directed mutagenesis of the mRNA greatly reduced translation pausing without altering the polypeptide sequence, demonstrating unambiguously a role for this mRNA sequence in translation pausing. SR alpha polypeptides synthesized from the non-pausing mRNA were impaired in co-translational membrane anchoring. Furthermore, co-translational membrane assembly of SR alpha appears to anchor polysomes translating SR alpha to membranes.     

Enhancement of membrane insertion and function in a type IIIb membrane protein following introduction of a cleavable signal peptide.

The human beta 2 adrenergic receptor is a type IIIb membrane protein. It has a putative seven-transmembrane topology but lacks an amino-terminal cleavable signal sequence. The mechanism by which the amino terminus of the beta 2 receptor is translocated across the endoplasmic reticulum membrane is unknown. Furthermore, it is not known if translocation as a type IIIb protein is essential for the proper folding. Our studies indicate that conversion of beta 2 receptor from a type IIIb to a type IIIa membrane protein by introducing an NH2-terminal cleavable signal sequence enhances translocation of the receptor into the endoplasmic reticulum membrane, thereby facilitating expression of functional receptor.