The C-terminal domain of TRPV4 is essential for plasma membrane localization

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.     

The C-terminus is critical for the functional expression of the human serotonin transporter

The plasma membrane serotonin transporter (SERT) has an important role in terminating serotonergic neurotransmission by re-uptake of 5-HT from the synaptic cleft. The expression of SERT on the cell surface is therefore a critical factor. In this study, we examined the role of the carboxyl terminus of SERT in trafficking to the plasma membrane. 5-HT uptake activity was used to measure the effects of systematic deletions or alanine substitutions in the C-terminus. We found that deletion of 16 amino acids in the distal C-terminus had no effect on uptake activity, whereas further deletion was detrimental for the function of SERT. Cell surface biotinylation was used to determine the role of the C-terminus in localization and trafficking. We showed that the C-terminus is crucial for the delivery of SERT to the plasma membrane and that the deletion of this part of the transporter results in a lack of mature glycosylation and impaired trafficking to the plasma membrane. Furthermore, the C-terminally truncated mutants were shown to have a dominant negative effect on wild-type SERT uptake activity.     

Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein

Elongation of very long chain fatty acids 4 (ELOVL4) is a novel member of the ELO family of genes that are involved in fatty acid metabolism. ELOVL4 encodes a putative transmembrane protein of 314 amino acids that carries a possible endoplasmic reticulum (ER) retention/retrieval signal (KXKXX) at the C-terminus. Two distinct mutations, a 5-bp deletion and a complex mutation from the same region in exon 6 of this gene, have been reported so far and are associated with autosomal dominant atrophic macular degeneration (adMD/STGD3). Both of these deletions could result in C-terminal truncation and loss of the ER retention signal in the mutant protein. We expressed the wild-type and mutant proteins in COS-7 and CHO cells to study the intracellular distribution of ELOVL4 and to identify possible implications of the above mutations in its localization. Immunofluorescence analysis of these proteins along with organelle marker antibodies revealed predominant ER localization for wild-type ELOVL4. Targeted deletion of the dilysine motif at the C-terminus of the protein resulted in the loss of ER localization. Immunoelectron microscopy and immunofluorescence analysis revealed a similar ER localization pattern for the protein in human photoreceptors. These data indicate that ELOVL4 is an ER-resident protein, which supports its suggested function in fatty acid elongation. We also demonstrate that the localization of both mutant proteins was dramatically changed from an ER to a Golgi distribution. Our observations suggest that the consequences of defective protein trafficking could underlie the molecular mechanism associated with degeneration of the macula in the patients with adMD/STGD3.     

Cytoplasmic and transmembrane domain deletions of Na,K-ATPase beta-subunit. Effects on subunit assembly and intracellular transport.

cDNAs encoding Na,K-ATPase beta-subunits containing deletions in the cytoplasmic domain or in the single membrane-spanning domain of the molecule were constructed and expressed in mouse L cells to determine the effect(s) of deletions in these domains on alpha/beta-subunit assembly and intracellular targeting. Avian beta-subunits lacking some or all of the cytoplasmic domain (endodomain) assemble with the endogenous mouse alpha-subunit and are correctly transported to the plasma membrane. Mutants containing deletions in the transmembrane domain were constructed by fusing portions of cDNAs encoding the amino-terminal one-third of human beta-subunit deletion mutants with avian beta-subunit cDNA encoding the carboxyl two-thirds of the molecule. A deletion of 3 amino acids in transmembrane domain resulted in correct alpha/beta-subunit assembly and localization to the plasma membrane. In contrast, deletions of 5 or more amino acids in the transmembrane domain prevented expression of the beta-subunit at the cell surface and resulted in the accumulation of these molecules in the ER. In spite of these targeting differences, all beta-subunit mutants capable of membrane insertion were also able to assemble with the alpha-subunit. These results suggest that the specificity for alpha/beta assembly resides in the ectodomains of the subunits.     

Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3

The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3‐transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists‐activated Ca²⁺ influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein‐protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes.     

Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.     

Identification of a Membrane-targeting Domain of the Transient Receptor Potential Canonical (TRPC)4 Channel Unrelated to Its Formation of a Tetrameric Structure

Canonical transient receptor potential (TRPC) channels are Ca²⁺-permeable nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). The TRPC4 channel is expressed in a punctate distribution in the membrane. To identify the regulating region of the channel trafficking to the membrane, we generated deletion mutants of the TRPC4 channel. We determined that when either region that was downstream of the 20 amino acids of the N terminus or the 700–730 amino acids was deleted, the mutants were retained in the endoplasmic reticulum. By coexpression of the wild-type TRPC4 with deletion mutants, we found that the 23–29 amino acids of the N terminus regulate a membrane trafficking. Additionally, by the fluorescence resonance energy transfer (FRET) method, we found that the regions downstream of the 99 amino acid region of the N terminus and upstream of the 730 amino acid region in the C terminus produce assembly of the TRPC4 tetramers. We inferred the candidate proteins that regulate or interact with the 23–29 domain of TRPC4.     

Effect of block deletions in the C-terminus on the functional expression of human anion exchanger 1 (AE1)

The human anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells and is responsible for the exchange of Cl(-) for HCO(3)(-). However, the detailed role played by the AE1 C-terminal region in the anion translocation and membrane trafficking process remains unclear. In this paper, we created four mutants in the human AE1 C-terminus by deletion of the residues Ala(891)-Phe(895), Asp(896)-Glu(899), Asp(902)-Glu(906) and Val(907)-Val(911), to investigate the role of these sequences in functional expression of AE1. WT AE1 and its deletion mutant constructs were expressed in HEK 293 cells. Western blotting showed that deletions of Ala(891)-Phe(895), Asp(896)-Glu(899), and Val(907)-Val(911) induced high expression of AE1, whereas loss of Asp(902)-Glu(906) results in stable low expression. Pulse chase assays of WT AE1 and its mutants showed that the stability of protein is unaffected by the levels of expression of the AE1 and its mutants. Ala(891)-Phe(895), Asp(902)-Glu(906) and Val(907)-Val(911) mutants exhibited lower levels of trafficking to the plasma membrane compared with WT AE1, while the Asp(896)-Glu(899) mutant was more highly expressed at the plasma membrane. The decreased ability of the mutants to mediate Cl(-)/HCO(3)(-) exchange in transfected cells revealed that the deletion sequences have an important role in transport activity. These results demonstrate that the studied residues in the AE1 C-terminus differently affect the expression, membrane trafficking and functional folding of AE1.     

Domain requirement for the membrane trafficking and targeting of syntaxin 1A

Syntaxin plays a key role in intracellular membrane fusion in eukaryotic cells. The function of syntaxin relies on its proper trafficking to and targeting at the target membrane. The mechanisms underlying the trafficking and targeting of syntaxin to its physiological sites remain poorly understood. Here we have analyzed the trafficking of syntaxin 1A in INS-1 and CHO cells. We have identified the transmembrane domain together with several flanking positive-charged amino acids as the minimal domain required for the membrane delivery. Interestingly, we found that SNARE motif-exposed syntaxin 1A mutants were retained in endoplasmic reticulum (ER) and failed to transport to the cell surface in the absence of SNAP-25, suggesting that the exposure of the SNARE motif causes ER retention and complexation with SNAP-25 helps the ER escape. Finally, our data propose two key roles for the H(abc) domain: to protect nonspecific interaction by masking the SNARE motif and to participate in the clustering of syntaxin 1A to the fusion sites in the plasma membrane.     

Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.     

Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells

ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N-terminus (residues 1-234) is a GTPase activating protein (GAP) for RhoGTPases, while the C-terminus (residues 232-453) encodes an ADP-ribosyltransferase. Utilizing a series of N-terminal deletion peptides of ExoS and an epitope-tagged full-length ExoS, two independent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N-terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90-234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP-ribosyltransferase activities.     

Role of C-terminus of Kir7.1 potassium channel in cell-surface expression

Inward rectifier K+ channel Kir7.1 is predominantly expressed on the plasma membrane of a variety of ion-transporting epithelia. The electrophysiological property of Kir7.1 has been well characterized but the mechanism underlying the plasma-membrane targeting remains elusive. To address this issue, we examined the effect of deletion and site-directed mutagenesis on the plasma-membrane localization of Kir7.1 in Madin-Darby canine kidney cells by immunofluorescence microscopy and cell-surface biotinylation. Although deletions of up to 37 amino acid residues from the C-terminus had no effect, further deletion resulted in accumulation of the mutant proteins in intracellular membranes. No sequence motif for subcellular targeting was found in the distal C-terminal region. The cell-surface expression of the deletion mutant lacking 38 or 40 C-terminal residues was restored by addition of one or three alanine residues, respectively, to the C-terminus end. These results suggest that the C-terminal length plays an important role in the plasma-membrane localization of Kir7.1.     

The retention factor p11 confers an endoplasmic reticulum-localization signal to the potassium channel TASK-1

The interaction of the adaptor protein p11, also denoted S100A10, with the C-terminus of the two-pore-domain K+ channel TASK-1 was studied using yeast two-hybrid analysis, glutathione S-transferase pull-down, and co-immunoprecipitation. We found that p11 interacts with a 40 amino-acid region in the proximal C-terminus of the channel. In heterologous expression systems, deletion of the p11-interacting domain enhanced surface expression of TASK-1. Attachment of the p11-interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C-terminal end of p11. Imaging of EGFP-tagged TASK-1 channels in COS cells suggested that wild-type TASK-1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK-1 to the surface membrane. Our results suggest that binding of p11 to TASK-1 retards the surface expression of the channel, most likely by virtue of a di-lysine retention signal at the C-terminus of p11. Thus, the cytosolic protein p11 may represent a 'retention factor' that causes localization of the channel to the ER.     

PACSINs bind to the TRPV4 cation channel. PACSIN 3 modulates the subcellular localization of TRPV4

TRPV4 is a cation channel that responds to a variety of stimuli including mechanical forces, temperature, and ligand binding. We set out to identify TRPV4-interacting proteins by performing yeast two-hybrid screens, and we isolated with the avian TRPV4 amino terminus the chicken orthologues of mammalian PACSINs 1 and 3. The PACSINs are a protein family consisting of three members that have been implicated in synaptic vesicular membrane trafficking and regulation of dynamin-mediated endocytotic processes. In biochemical interaction assays we found that all three murine PACSIN isoforms can bind to the amino terminus of rodent TRPV4. No member of the PACSIN protein family was able to biochemically interact with TRPV1 and TRPV2. Co-expression of PACSIN 3, but not PACSINs 1 and 2, shifted the ratio of plasma membrane-associated versus cytosolic TRPV4 toward an apparent increase of plasma membrane-associated TRPV4 protein. A similar shift was also observable when we blocked dynamin-mediated endocytotic processes, suggesting that PACSIN 3 specifically affects the endocytosis of TRPV4, thereby modulating the subcellular localization of the ion channel. Mutational analysis shows that the interaction of the two proteins requires both a TRPV4-specific proline-rich domain upstream of the ankyrin repeats of the channel and the carboxyl-terminal Src homology 3 domain of PACSIN 3. Such a functional interaction could be important in cell types that show distribution of both proteins to the same subcellular regions such as renal tubule cells where the proteins are associated with the luminal plasma membrane.     

The cytoplasmic C-terminus of the sulfonylurea receptor is important for KATP channel function but is not key for complex assembly or trafficking.

ATP-sensitive K+ channels are an octameric assembly of two proteins, a sulfonylurea receptor (SUR1) and an ion conducting subunit (Kir 6.0). We have examined the role of the C-terminus of SUR1 by expressing a series of truncation mutants together with Kir6.2 stably in HEK293 cells. Biochemical analyses using coimmunoprecipitation indicate that SUR1 deletion mutants and Kir6.2 assemble and that a SUR1 deletion mutant binds glibenclamide with high affinity. Electrophysiological recordings indicate that ATP sensitivity is normal but the response of the mutant channel complexes to tolbutamide, MgADP and diazoxide is disturbed. Quantitative immunofluorescence and cell surface biotinylation supports the idea that there is little disturbance in the efficiency of trafficking. Our data show that deletions of the C-terminal most cytoplasmic domain of SUR1, can result in functional channels at the plasma membrane in mammalian cells that have an abnormal response to physiological and pharmacological agents.     

The role of the N terminus and transmembrane domain of TRPM8 in channel localization and tetramerization

Transient receptor potential (TRP) channels are a family of cation channels involved in diverse cellular functions. They are composed of a transmembrane domain of six putative transmembrane segments flanked by large N- and C-terminal cytoplasmic domains. The melastatin subfamily (TRPM) channels have N-terminal domains of approximately 700 amino acids with four regions of shared homology and C-terminal domains containing the conserved TRP domain followed by a coiled-coil region. Here we investigated the effects of N- and C-terminal deletions on the cold and menthol receptor, TRPM8, expressed heterologously in Sf21 insect cells. Patch-clamp electrophysiology was used to study channel activity and revealed that only deletion of the first 39 amino acids was tolerated by the channel. Further N-terminal truncation or any C-terminal deletions prevented proper TRPM8 function. Confocal microscopy with immunofluorescence revealed that amino acids 40-86 are required for localization to the plasma membrane. Furthermore, analysis of deletion mutant oligomerization shows that the transmembrane domain is sufficient for TPRM8 assembly into tetramers. TRPM8 channels with C-terminal deletions tetramerize and localize properly but are inactive, indicating that although not essential for tetramerization and localization, the C terminus is critical for proper function of the channel sensor and/or gate.     

Homo- and hetero-dimeric architecture of the human liver Na⁺-dependent taurocholate co-transporting protein

The NTCP (Na?-taurocholate co-transporting protein)/SLC10A [solute carrier family 10 (Nav/bile acid co-transporter family)] 1 is tightly controlled to ensure hepatic bile salt uptake while preventing toxic bile salt accumulation. Many transport proteins require oligomerization for their activity and regulation. This is not yet established for bile salt transporters. The present study was conducted to elucidate the oligomeric state of NTCP. Chemical cross-linking revealed the presence of NTCP dimers in rat liver membranes and U2OS cells stably expressing NTCP. Co-immunoprecipitation of tagged NTCP proteins revealed a physical interaction between subunits. The C-terminus of NTCP was not required for subunit interaction, but was essential for exit from the ER (endoplasmic reticulum). NTCP without its C-terminus (NTCP Y307X) retained full-length wtNTCP (wild-type NTCP) in the ER in a dominant fashion, suggesting that dimerization occurs early in the secretory pathway. FRET (fluorescence resonance energy transfer) using fluorescently labelled subunits further demonstrated that dimerization persists at the plasma membrane. NTCP belongs to the SLC10A protein family which consists of seven members. NTCP co-localized in U2OS cells with SLC10A4 and SLC10A6, but not with SLC10A3, SLC10A5 or SLC10A7. SLC10A4 and SLC10A6 co-immunoprecipitated with NTCP, demonstrating that heteromeric complexes can be formed between SLC10A family members in vitro. Expression of SLC10A4 and NTCP Y307X resulted in a reduction of NTCP abundance at the plasma membrane and NTCP-mediated taurocholate uptake, whereas expression of SLC10A6 or NTCP E257N, an inactive mutant, did not affect NTCP function. In conclusion, NTCP adopts a dimeric structure in which individual subunits are functional. Bile salt uptake is influenced by heterodimerization when this impairs NTCP plasma membrane trafficking.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

In Vitro Mutational Analysis of the β2 Adrenergic Receptor,an In Vivo Surrogate Odorant Receptor

Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs.