Evolutionarily conserved networks of residues mediate allosteric communication in proteins

A fundamental goal in cellular signaling is to understand allosteric communication, the process by which signals originating at one site in a protein propagate reliably to affect distant functional sites. The general principles of protein structure that underlie this process remain unknown. Here, we describe a sequence-based statistical method for quantitatively mapping the global network of amino acid interactions in a protein. Application of this method for three structurally and functionally distinct protein families (G protein-coupled receptors, the chymotrypsin class of serine proteases and hemoglobins) reveals a surprisingly simple architecture for amino acid interactions in each protein family: a small subset of residues forms physically connected networks that link distant functional sites in the tertiary structure. Although small in number, residues comprising the network show excellent correlation with the large body of mechanistic data available for each family. The data suggest that evolutionarily conserved sparse networks of amino acid interactions represent structural motifs for allosteric communication in proteins.     

A machine learning based method for the prediction of G protein-coupled receptor-binding PDZ domain proteins

G protein-coupled receptors (GPCRs) are part of multi-protein networks called ‘receptosomes’. These GPCR interacting proteins (GIPs) in the receptosomes control the targeting, trafficking and signaling of GPCRs. PDZ domain proteins constitute the largest protein family among the GIPs, and the predominant function of the PDZ domain proteins is to assemble signaling pathway components into close proximity by recognition of the last four C-terminal amino acids of GPCRs. We present here a machine learning based approach for the identification of GPCR-binding PDZ domain proteins. In order to characterize the network of interactions between amino acid residues that contribute to the stability of the PDZ domain-ligand complex and to encode the complex into a feature vector, amino acid contact matrices and physicochemical distance matrix were constructed and adopted. This novel machine learning based method displayed high performance for the identification of PDZ domain-ligand interactions and allowed the identification of novel GPCR-PDZ domain protein interactions.     

Dimerization in aminergic G-protein-coupled receptors: application of a hidden-site class model of evolution

G-Protein-coupled receptors (GPCRs) are an important superfamily of transmembrane proteins involved in cellular communication. Recently, it has been shown that dimerization is a widely occurring phenomenon in the GPCR superfamily, with likely important physiological roles. Here we use a novel hidden-site class model of evolution as a sequence analysis tool to predict possible dimerization interfaces in GPCRs. This model aims to simulate the evolution of proteins at the amino acid level, allowing the analysis of their sequences in an explicitly evolutionary context. Applying this model to aminergic GPCR sequences, we first validate the general reasoning behind the model. We then use the model to perform a family specific analysis of GPCRs. Accounting for the family structure of these proteins, this approach detects different evolutionarily conserved and accessible patches on transmembrane (TM) helices 4-6 in different families. On the basis of these findings, we propose an experimentally testable dimerization mechanism, involving interactions among different combinations of these helices in different families of aminergic GPCRs.     

Change in allosteric network affects binding affinities of PDZ domains: analysis through perturbation response scanning

The allosteric mechanism plays a key role in cellular functions of several PDZ domain proteins (PDZs) and is directly linked to pharmaceutical applications; however, it is a challenge to elaborate the nature and extent of these allosteric interactions. One solution to this problem is to explore the dynamics of PDZs, which may provide insights about how intramolecular communication occurs within a single domain. Here, we develop an advancement of perturbation response scanning (PRS) that couples elastic network models with linear response theory (LRT) to predict key residues in allosteric transitions of the two most studied PDZs (PSD-95 PDZ3 domain and hPTP1E PDZ2 domain). With PRS, we first identify the residues that give the highest mean square fluctuation response upon perturbing the binding sites. Strikingly, we observe that the residues with the highest mean square fluctuation response agree with experimentally determined residues involved in allosteric transitions. Second, we construct the allosteric pathways by linking the residues giving the same directional response upon perturbation of the binding sites. The predicted intramolecular communication pathways reveal that PSD-95 and hPTP1E have different pathways through the dynamic coupling of different residue pairs. Moreover, our analysis provides a molecular understanding of experimentally observed hidden allostery of PSD-95. We show that removing the distal third alpha helix from the binding site alters the allosteric pathway and decreases the binding affinity. Overall, these results indicate that (i) dynamics plays a key role in allosteric regulations of PDZs, (ii) the local changes in the residue interactions can lead to significant changes in the dynamics of allosteric regulations, and (iii) this might be the mechanism that each PDZ uses to tailor their binding specificities regulation.     

Orthosteric and allosteric binding sites of P2X receptors

P2X receptors for ATP comprise a distinct family of ligand gated ion channels with a range of properties. They have been shown to be involved in a variety of physiological processes including blood clotting, sensory perception, pain sensation, bone formation as well as inflammation and may provide a number of novel drug targets. In addition to the orthosteric site for ATP binding it has been suggested that there may be additional allosteric sites that regulate agonist action at the receptor. There is currently no crystal structure available for P2X receptors and the lack of sequence similarity to other ATP binding proteins has meant that a mutagenesis-based approach has been used primarily to investigate receptor structure-function. This review aims to provide an overview of recent work that gives an insight into residues involved in ATP action and allosteric regulation.     

Allosteric Effects of Sodium Ion Binding on Activation of the M3 Muscarinic G-Protein-Coupled Receptor

G-protein-coupled receptors (GPCRs) are important membrane proteins that mediate cellular signaling and represent primary targets for about one-third of currently marketed drugs. Recent x-ray crystallographic studies identified distinct conformations of GPCRs in the active and inactive states. An allosteric sodium ion was found bound to a highly conserved D2.50 residue in inactive GPCRs, whereas the D2.50 allosteric pocket became collapsed in active GPCR structures. However, the dynamic mechanisms underlying these observations remain elusive. In this study, we aimed to understand the mechanistic effects of sodium ion binding on dynamic activation of the M3 muscarinic GPCR through long-timescale accelerated molecular dynamics (aMD) simulations. Results showed that with the D2.50 residue deprotonated, the M3 receptor is bound by an allosteric sodium ion and confined mostly in the inactive state with remarkably reduced flexibility. In contrast, the D2.50-protonated receptor does not exhibit sodium ion binding to the D2.50 allosteric site and samples a significantly larger conformational space. The receptor activation is captured and characterized by large-scale structural rearrangements of the transmembrane helices via dynamic hydrogen bond and salt bridge interactions. The residue motions are highly correlated during receptor activation. Further network analysis revealed that the allosteric signaling between residue D2.50 and key residues in the intracellular, extracellular, and orthosteric pockets is significantly weakened upon sodium ion binding.     

Evolutionary analysis of rhodopsin and cone pigments: connecting the three-dimensional structure with spectral tuning and signal transfer

Extensive sequence data and structural sampling of expressed proteins from different species lead to the idea that entire molecules or specific domain folds belong to large superfamilies of proteins. A subset of G protein-coupled receptors, one of the largest families involved in cellular signaling, rod and cone opsins are involved in phototransduction in photoreceptor cells. Here, the evolutionary analysis of opsin sequences and structures predicts key residues involved in the transmission of the signal from the binding site of the chromophore to the cytoplasmic surface and residues that are involved in the spectral tuning of opsins to short wavelengths of light.     

Allostery in GPCRs: 'MWC' revisited

G protein-coupled receptors (GPCRs) constitute the largest family of receptors in the genome and are the targets for at least 30% of current medicines. In recent years, there has been a dramatic increase in the discovery of allosteric modulators of GPCR activity and a growing appreciation of the diverse modes by which GPCRs can be regulated by both orthosteric and allosteric ligands. Interestingly, some of the contemporary views of GPCR function reflect characteristics that are shared by prototypical allosteric proteins, as encompassed in the classic Monod-Wyman-Changeux (MWC) model initially proposed for enzymes and subsequently extended to other protein families. In this review, we revisit the MWC model in the context of emerging structural, functional and operational data on GPCR allostery.     

On the accuracy of inferring energetic coupling between distant sites in protein families from evolutionary imprints: Illustrations using lattice model

It is suspected that correlated motions among a subset of spatially separated residues drive conformational dynamics not only in multidomain but also in single domain proteins. Sequence and structure‐based methods have been proposed to determine covariation between two sites on a protein. The statistical coupling analysis (SCA) that compares the changes in probability at two sites in a multiple sequence alignment (MSA) and a subset of the MSA has been used to infer the network of residues that encodes allosteric signals in protein families. The structural perturbation method (SPM), that probes the response of a local perturbation at all other sites, has been used to probe the allostery wiring diagram in biological machines and enzymes. To assess the efficacy of the SCA, we used an exactly soluble two dimensional lattice model and performed double‐mutant cycle (DMC) calculations to predict the extent of physical coupling between two sites. The predictions of the SCA and the DMC results show that only residues that are in contact in the native state are accurately identified. In addition, covariations among strongly interacting residues are most easily identified by the SCA. These conclusions are consistent with the DMC experiments on the PDZ family. Good correlation between the SCA and the DMC is only obtained by performing multiple experiments that vary the nature of amino acids at a given site. In contrast, the energetic coupling found in experiments for the PDZ domain are recovered using the SPM. We also predict, using the SPM, several residues that are coupled energetically. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Novel Allosteric Modulators of G Protein-coupled Receptors

G protein-coupled receptors (GPCRs) are allosteric proteins, because their signal transduction relies on interactions between topographically distinct, yet conformationally linked, domains. Much of the focus on GPCR allostery in the new millennium, however, has been on modes of targeting GPCR allosteric sites with chemical probes due to the potential for novel therapeutics. It is now apparent that some GPCRs possess more than one targetable allosteric site, in addition to a growing list of putative endogenous modulators. Advances in structural biology are also shedding new insights into mechanisms of allostery, although the complexities of candidate allosteric drugs necessitate rigorous biological characterization.     

Type I PDZ ligands are sufficient to promote rapid recycling of G Protein-coupled receptors independent of binding to N-ethylmaleimide-sensitive factor

Molecular sorting of G protein-coupled receptors (GPCRs) between divergent recycling and lysosomal pathways determines the functional consequences of agonist-induced endocytosis. The carboxyl-terminal cytoplasmic domain of the beta2 adrenergic receptor (beta2AR) mediates both PDZ binding to Na+/H+ exchanger regulatory factor/ezrin/radixin/moesin-binding phosphoprotein of 50 kDa (NHERF/EBP50) family proteins and non-PDZ binding to the N-ethylmaleimide-sensitive factor (NSF). We have investigated whether PDZ interaction(s) are actually sufficient to promote rapid recycling of endocytosed receptors and, if so, whether PDZ-mediated sorting is restricted to the beta2AR tail or to sequences that bind NHERF/EBP50. The trafficking effects of short (10 residue) sequences differing in PDZ and NSF binding properties were examined using chimeric mutant receptors. The recycling activity of the beta2AR-derived tail sequence was not blocked by a point mutation that selectively disrupts binding to NSF, and naturally occurring PDZ ligand sequences were identified that do not bind detectably to NSF yet function as strong recycling signals. The carboxyl-terminal cytoplasmic domain of the beta1-adrenergic receptor, which does not bind either to NSF or NHERF/EBP50 and interacts selectively with a distinct group of PDZ proteins, promoted rapid recycling of chimeric mutant receptors with efficiency similarly high as that of the beta2AR tail. These results indicate that PDZ domain-mediated protein interactions are sufficient to promote rapid recycling of GPCRs, independent of binding to NSF. They also suggest that PDZ-directed recycling is a rather general mechanism of GPCR regulation, which is not restricted to a single GPCR, and may involve additional PDZ domain-containing protein(s) besides NHERF/EBP50.     

Quantitative analysis of allosteric drug-protein binding by biointeraction chromatography

Allosteric interactions are important in many biological processes. They occur when the interactions of one substance with a binding agent changes the interactions of a second substance with the same agent at a separate site. Such interactions are often observed during the binding of drugs to blood proteins such as human serum albumin (HSA). Most previous studies of allosteric interactions have involved only qualitative observations of increased or decreased binding. In this study, we present an approach for quantitatively characterizing such allosteric effects using protein columns. The method is used to examine the interactions of ibuprofen/S-lorazepam acetate, S-oxazepam hemisuccinate/R-oxazepam hemisuccinate, and L-tryptophan/phenytoin during their binding to HSA. This approach can be applied to other receptors or biopolymers and can be used to independently examine the effects of two competing agents during an allosteric interaction.     

Prediction of the odorant binding site of olfactory receptor proteins by human-mouse comparisons

Olfactory receptors (ORs) are a large family of proteins involved in the recognition and discrimination of numerous odorants. These receptors belong to the G-protein coupled receptor (GPCR) hyperfamily, for which little structural data are available. In this study we predict the binding site residues of OR proteins by analyzing a set of 1441 OR protein sequences from mouse and human. The central insight utilized is that functional contact residues would be conserved among pairs of orthologous receptors, but considerably less conserved among paralogous pairs. Using judiciously selected subsets of 218 ortholog pairs and 518 paralog pairs, we have identified 22 sequence positions that are both highly conserved among the putative orthologs and variable among paralogs. These residues are disposed on transmembrane helices 2 to 7, and on the second extracellular loop of the receptor. Strikingly, although the prediction makes no assumption about the location of the binding site, these amino acid positions are clustered around a pocket in a structural homology model of ORs, mostly facing the inner lumen. We propose that the identified positions constitute the odorant binding site. This conclusion is supported by the observation that all but one of the predicted binding site residues correspond to ligand-contact positions in other rhodopsin-like GPCRs.     

基于弹性网络模型的蛋白质变构路径与关键残基识别研究

目的 变构效应在蛋白质生物学功能执行过程中发挥着重要的调控作用，如何基于蛋白质空间结构，有效识别变构信号的传播路径和关键的残基位点是蛋白质结构-功能关系研究领域的热点科学问题。方法 本研究利用基于弹性网络模型（elastic network model，ENM）的力分布计算方法，通过分析蛋白质对外力的响应过程，来识别体系的变构路径以及变构过程中的关键残基。在该方法中，对蛋白质的关键变构位点施加外力，通过对体系形变以及内力分布情况的分析，有效识别与外力承载区域形变相耦合的关键残基，从而得到力信号在蛋白质结构内的传播路径。结果 利用该方法研究了人类磷酸甘油酸激酶（human phosphoglycerate kinase，hPGK）和蛋白质酪氨酸磷酸酶（protein tyrosine phosphatase，PTP）PDZ2结构域的变构调控路径和关键残基。对于hPGK，识别出从底物结合位点到铰链区的两条变构信号传导路径。对于PTP PDZ2，也成功识别出从配体结合位点传递到蛋白质远端的两条长程变构调控路径。计算结果与实验和分子动力学（molecular dynamics，MD）模拟得到的结果一致。结论 本研究为蛋白质体系关键残基识别及变构路径研究提供了有效的分析方法。     

Distinct ligand specificity of the Tiam1 and Tiam2 PDZ domains

Guanine nucleotide exchange factor proteins of the Tiam family are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These proteins are modular and contain a variety of interaction domains, including a single post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Previous studies suggest that the specificities of the Tiam1 and Tiam2 PDZ domains are distinct. Here, we sought to conclusively define these specificities and determine their molecular origin. Using a combinatorial peptide library, we identified a consensus binding sequence for each PDZ domain. Analysis of these consensus sequences and binding assays with peptides derived from native proteins indicated that these two PDZ domains have overlapping but distinct specificities. We also identified residues in two regions (S(0) and S(-2) pockets) of the Tiam1 PDZ domain that are important determinants of ligand specificity. Site-directed mutagenesis of four nonconserved residues in these two regions along with peptide binding analyses confirmed that these residues are crucial for ligand affinity and specificity. Furthermore, double mutant cycle analysis of each region revealed energetic couplings that were dependent on the ligand being investigated. Remarkably, a Tiam1 PDZ domain quadruple mutant had the same specificity as the Tiam2 PDZ domain. Finally, analysis of Tiam family PDZ domain sequences indicated that the PDZ domains segregate into four distinct families based on the residues studied here. Collectively, our data suggest that Tiam family proteins have highly evolved PDZ domain-ligand interfaces with distinct specificities and that they have disparate PDZ domain-dependent biological functions.     

Integration of Evolutionary Features for the Identification of Functionally Important Residues in Major Facilitator Superfamily Transporters

The identification of functionally important residues is an important challenge for understanding the molecular mechanisms of proteins. Membrane protein transporters operate two-state allosteric conformational changes using functionally important cooperative residues that mediate long-range communication from the substrate binding site to the translocation pathway. In this study, we identified functionally important cooperative residues of membrane protein transporters by integrating sequence conservation and co-evolutionary information. A newly derived evolutionary feature, the co-evolutionary coupling number, was introduced to measure the connectivity of co-evolving residue pairs and was integrated with the sequence conservation score. We tested this method on three Major Facilitator Superfamily (MFS) transporters, LacY, GlpT, and EmrD. MFS transporters are an important family of membrane protein transporters, which utilize diverse substrates, catalyze different modes of transport using unique combinations of functional residues, and have enough characterized functional residues to validate the performance of our method. We found that the conserved cores of evolutionarily coupled residues are involved in specific substrate recognition and translocation of MFS transporters. Furthermore, a subset of the residues forms an interaction network connecting functional sites in the protein structure. We also confirmed that our method is effective on other membrane protein transporters. Our results provide insight into the location of functional residues important for the molecular mechanisms of membrane protein transporters.     

Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.     

Convergent Transmission of RNAi Guide-Target Mismatch Information across Argonaute Internal Allosteric Network

In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand “seed region” have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of a large number of internal allosteric pathways.     

Biochemical Large-Scale Interaction Analysis of Murine Olfactory Receptors and Associated Signaling Proteins with Post-Synaptic Density 95, Drosophila Discs Large,Zona-Occludens 1 (PDZ) Domains

G protein-coupled receptors (GPCRs) constitute the largest family among mammalian membrane proteins and are capable of initiating numerous essential signaling cascades. Various GPCR-mediated pathways are organized into protein microdomains that can be orchestrated and regulated through scaffolding proteins, such as PSD-95/discs-large/ZO1 (PDZ) domain proteins. However, detailed binding characteristics of PDZ–GPCR interactions remain elusive because these interactions seem to be more complex than previously thought. To address this issue, we analyzed binding modalities using our established model system. This system includes the 13 individual PDZ domains of the multiple PDZ domain protein 1 (MUPP1; the largest PDZ protein), a broad range of murine olfactory receptors (a multifaceted gene cluster within the family of GPCRs), and associated olfactory signaling proteins. These proteins were analyzed in a large-scale peptide microarray approach and continuative interaction studies. As a result, we demonstrate that canonical binding motifs were not overrepresented among the interaction partners of MUPP1. Furthermore, C-terminal phosphorylation and distinct amino acid replacements abolished PDZ binding promiscuity. In addition to the described in vitro experiments, we identified new interaction partners within the murine olfactory epithelium using pull-down-based interactomics and could verify the partners through co-immunoprecipitation. In summary, the present study provides important insight into the complexity of the binding characteristics of PDZ–GPCR interactions based on olfactory signaling proteins, which could identify novel clinical targets for GPCR-associated diseases in the future.PDZ domain proteins comprise one of the largest families among interaction domain scaffolding proteins and are highly abundant in various multicellular eukaryotic species. These proteins fulfill important physiological functions in a broad range of different tissues and cells as they orchestrate complex protein networks. Among putative PDZ interaction partners, one important protein family is the group of GPCRs¹, constituting the largest family of membrane proteins in mammals (1). Here, signal efficiency, speed, desensitization, and internalization can be modulated by PDZ proteins (2–5). Olfactory receptors (ORs) represent a multigene family within this group of seven-transmembrane domain proteins and encompass 2% of the mammalian genome (6). Belonging to class I GPCRs, ORs share many general features of this receptor family, making them an interesting target for interactions involving PDZ proteins. Until recently, an organizing complex builder, such as the inactivation no afterpotential D (InaD) protein in the visual system of Drosophila melanogaster (7, 8), could not be clearly identified for olfactory signaling.The multiple PDZ domain protein 1, with 13 individual PDZ domains, represents the largest of the described PDZ proteins to date (9) and interacts with different GPCRs (10–12). One well-described example is its interaction with GABA_B receptors, leading to enhanced receptor stability at the plasma membrane and prolonged signaling duration (2). In previous studies, we demonstrated that PDZ domains 1 + 2 can interact with a selected subset of ORs (13). Furthermore, we showed that MUPP1 binds to a specific OR and that most of the described proteins are involved in mammalian olfactory signal transduction in the native system, making MUPP1 a promising candidate for orchestrating the olfactory system (14).Many PDZ–ligand interactions depend on classical binding motifs at the ligand''s C-terminal end, thereby building weak transient protein complexes (15, 16). However, an increasing number of PDZ interactions have emerged that seem to provide more complex binding modalities, differing from the canonical interactions (17, 18). Ligand binding seems not to be exclusively restricted to C-terminal sites, and PDZ domains cannot be distinctly classified but are evenly distributed throughout a selective space (17, 19–21). Therefore, it is of great interest to analyze OR–PDZ interactions to characterize the putative binding requirements and to further investigate the role of MUPP1 in olfactory signaling.In the present study, we characterized the binding modalities between the 13 individual PDZ domains of MUPP1 and a broad range of murine olfactory receptors in a large-scale approach, indicating that classical binding motifs were not overrepresented among the evaluated binding partners. In addition, we identified new binding partners from the murine olfactory epithelium using pull-down-based interactomics.     

Spatially Restricted G Protein-coupled Receptor Activity via Divergent Endocytic Compartments

Postendocytic sorting of G protein-coupled receptors (GPCRs) is driven by their interactions between highly diverse receptor sequence motifs with their interacting proteins, such as postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) (PDZ) domain proteins. However, whether these diverse interactions provide an underlying functional specificity, in addition to driving sorting, is unknown. Here we identify GPCRs that recycle via distinct PDZ ligand/PDZ protein pairs that exploit their recycling machinery primarily for targeted endosomal localization and signaling specificity. The luteinizing hormone receptor (LHR) and β2-adrenergic receptor (B2AR), two GPCRs sorted to the regulated recycling pathway, underwent divergent trafficking to distinct endosomal compartments. Unlike B2AR, which traffics to early endosomes (EE), LHR internalizes to distinct pre-early endosomes (pre-EEs) for its recycling. Pre-EE localization required interactions of the LHR C-terminal tail with the PDZ protein GAIP-interacting protein C terminus, inhibiting its traffic to EEs. Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling. Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment. We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.