GoLoco Motif Research Articles

G-protein Signaling Modulator-3 Regulates Heterotrimeric G-protein Dynamics through Dual Association with Gβ and Gαi Protein Subunits

Regulation of the assembly and function of G-protein heterotrimers (Gα·GDP/Gβγ) is a complex process involving the participation of many accessory proteins. One of these regulators, GPSM3, is a member of a family of proteins containing one or more copies of a small regulatory motif known as the GoLoco (or GPR) motif. Although GPSM3 is known to bind Gα(i)·GDP subunits via its GoLoco motifs, here we report that GPSM3 also interacts with the Gβ subunits Gβ1 to Gβ4, independent of Gγ or Gα·GDP subunit interactions. Bimolecular fluorescence complementation studies suggest that the Gβ-GPSM3 complex is formed at, and transits through, the Golgi apparatus and also exists as a soluble complex in the cytoplasm. GPSM3 and Gβ co-localize endogenously in THP-1 cells at the plasma membrane and in a juxtanuclear compartment. We provide evidence that GPSM3 increases Gβ stability until formation of the Gβγ dimer, including association of the Gβ-GPSM3 complex with phosducin-like protein PhLP and T-complex protein 1 subunit eta (CCT7), two known chaperones of neosynthesized Gβ subunits. The Gβ interaction site within GPSM3 was mapped to a leucine-rich region proximal to the N-terminal side of its first GoLoco motif. Both Gβ and Gα(i)·GDP binding events are required for GPSM3 activity in inhibiting phospholipase-Cβ activation. GPSM3 is also shown in THP-1 cells to be important for Akt activation, a known Gβγ-dependent pathway. Discovery of a Gβ/GPSM3 interaction, independent of Gα·GDP and Gγ involvement, adds to the combinatorial complexity of the role of GPSM3 in heterotrimeric G-protein regulation.

Computational Design of the Sequence and Structure of a Protein-Binding Peptide

The de novo design of protein-binding peptides is challenging because it requires the identification of both a sequence and a backbone conformation favorable for binding. We used a computational strategy that iterates between structure and sequence optimization to redesign the C-terminal portion of the RGS14 GoLoco motif peptide so that it adopts a new conformation when bound to Gα(i1). An X-ray crystal structure of the redesigned complex closely matches the computational model, with a backbone root-mean-square deviation of 1.1 Å.

Structural Determinants of Affinity Enhancement between GoLoco Motifs and G-Protein α Subunit Mutants

GoLoco motif proteins bind to the inhibitory G(i) subclass of G-protein α subunits and slow the release of bound GDP; this interaction is considered critical to asymmetric cell division and neuro-epithelium and epithelial progenitor differentiation. To provide protein tools for interrogating the precise cellular role(s) of GoLoco motif/Gα(i) complexes, we have employed structure-based protein design strategies to predict gain-of-function mutations that increase GoLoco motif binding affinity. Here, we describe fluorescence polarization and isothermal titration calorimetry measurements showing three predicted Gα(i1) point mutations, E116L, Q147L, and E245L; each increases affinity for multiple GoLoco motifs. A component of this affinity enhancement results from a decreased rate of dissociation between the Gα mutants and GoLoco motifs. For Gα(i1)(Q147L), affinity enhancement was seen to be driven by favorable changes in binding enthalpy, despite reduced contributions from binding entropy. The crystal structure of Gα(i1)(Q147L) bound to the RGS14 GoLoco motif revealed disorder among three peptide residues surrounding a well defined Leu-147 side chain. Monte Carlo simulations of the peptide in this region showed a sampling of multiple backbone conformations in contrast to the wild-type complex. We conclude that mutation of Glu-147 to leucine creates a hydrophobic surface favorably buried upon GoLoco peptide binding, yet the hydrophobic Leu-147 also promotes flexibility among residues 511-513 of the RGS14 GoLoco peptide.

Nucleobindin 1 is a Calcium Regulated Guanine Nucleotide Dissociation Inhibitor of GαI1

Nucleobindin 1 (NUCB1) is a widely expressed multi-domain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and show that sNUCB1 exists as a Calcium binding dimer in solution and binding to Calcium causes conformational changes in sNUCB1. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α subunits. We show that dimeric sNUCB1 binds to Gαi1 and that calcium-binding inhibits the interaction. The binding of sNUCB1 to Gαi1 inhibits its basal rate of GDP release and slows its rate and extent of GTPγS uptake. Additionally, our tissue culture experiments show that sNUCB1 prevents receptor-mediated Gαi-dependent inhibition of adenylyl cyclase (AC). Thus, we conclude that sNUCB1 is a calcium dependent guanine-nucleotide dissociation inhibitor (GDI) for Gαi1. To our knowledge sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. We also show that the mechanism of GDI activity of sNUCB1 is unique and does not arise from the consensus Goloco motif found in RGS proteins. We propose that cytoplasmic NUCB1 might function to regulate heterotrimeric G protein trafficking and signalling.

Structural Determinants of Affinity Enhancement between GoLoco Motifs and G-Protein α Subunit Mutants

Structural Determinants of Affinity Enhancement between GoLoco Motifs and G-Protein α Subunit Mutants

Nucleobindin 1 Is a Calcium-regulated Guanine Nucleotide Dissociation Inhibitor of Gαi1

Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent calcium cations. Calcium binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α subunits. We show that dimeric calcium-free sNUCB1 binds to expressed Gα(i1) and that calcium binding inhibits the interaction. The binding of sNUCB1 to Gα(i1) inhibits its basal rate of GDP release and slows its rate and extent of GTPγS uptake. Additionally, our tissue culture experiments show that sNUCB1 prevents receptor-mediated Gα(i)-dependent inhibition of adenylyl cyclase. Thus, we conclude that sNUCB1 is a calcium-dependent guanine nucleotide dissociation inhibitor (GDI) for Gα(i1). To our knowledge, sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. We also show that the mechanism of GDI activity of sNUCB1 is unique and does not arise from the consensus GoLoco motif found in RGS proteins. We propose that cytoplasmic NUCB1 might function to regulate heterotrimeric G protein trafficking and G protein-coupled receptor-mediated signal transduction pathways.

Specificity in Computational Protein Design

A long-standing goal of computational protein design is to create proteins similar to those found in Nature. One motivation is to harness the exquisite functional capabilities of proteins for our own purposes. The extent of similarity between designed and natural proteins also reports on how faithfully our models represent the selective pressures that determine protein sequences. As the field of protein design shifts emphasis from reproducing native-like protein structure to function, it has become important that these models treat the notion of specificity in molecular interactions. Although specificity may, in some cases, be achieved by optimization of a desired protein in isolation, methods have been developed to address directly the desire for proteins that exhibit specific functions and interactions.

Receptor-regulated Interaction of Activator of G-protein Signaling-4 and Gαi

Activator of G-protein signaling-4 (AGS4), via its three G-protein regulatory motifs, is well positioned to modulate G-protein signal processing by virtue of its ability to bind Galpha(i)-GDP subunits free of Gbetagamma. Apart from initial observations on the biochemical activity of the G-protein regulatory motifs of AGS4, very little is known about the nature of the AGS4-G-protein interaction, how this interaction is regulated, or where the interaction takes place. As an initial approach to these questions, we evaluated the interaction of AGS4 with Galpha(i1) in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and Galpha(i1) reciprocally tagged with either Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) demonstrated saturable, specific BRET signals. BRET signals observed between AGS4-RLuc and Galpha(i1)-YFP were reduced by G-protein-coupled receptor activation, and this agonist-induced reduction in BRET was blocked by pertussis toxin. In addition, specific BRET signals were observed for AGS4-RLuc and alpha(2)-adrenergic receptor-Venus, which were Galpha(i)-dependent and reduced by agonist, indicating that AGS4-Galpha(i) complexes are receptor-proximal. These data suggest that AGS4-Galpha(i) complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation.

Computational design of second‐site suppressor mutations at protein–protein interfaces

The importance of a protein-protein interaction to a signaling pathway can be established by showing that amino acid mutations that weaken the interaction disrupt signaling, and that additional mutations that rescue the interaction recover signaling. Identifying rescue mutations, often referred to as second-site suppressor mutations, controls against scenarios in which the initial deleterious mutation inactivates the protein or disrupts alternative protein-protein interactions. Here, we test a structure-based protocol for identifying second-site suppressor mutations that is based on a strategy previously described by Kortemme and Baker. The molecular modeling software Rosetta is used to scan an interface for point mutations that are predicted to weaken binding but can be rescued by mutations on the partner protein. The protocol typically identifies three types of specificity switches: knob-in-to-hole redesigns, switching hydrophobic interactions to hydrogen bond interactions, and replacing polar interactions with nonpolar interactions. Computational predictions were tested with two separate protein complexes; the G-protein Galpha(i1) bound to the RGS14 GoLoco motif, and UbcH7 bound to the ubiquitin ligase E6AP. Eight designs were experimentally tested. Swapping a buried hydrophobic residue with a polar residue dramatically weakened binding affinities. In none of these cases were we able to identify compensating mutations that returned binding to wild-type affinity, highlighting the challenges inherent in designing buried hydrogen bond networks. The strongest specificity switches were a knob-in-to-hole design (20-fold) and the replacement of a charge-charge interaction with nonpolar interactions (55-fold). In two cases, specificity was further tuned by including mutations distant from the initial design. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

GoLoco motif proteins binding to Gαi1: insights from molecular simulations

Molecular dynamics simulations, computational alanine scanning and sequence analysis were used to investigate the structural properties of the Gαi1/GoLoco peptide complex. Using these methodologies, binding of the GoLoco motif peptide to the Gαi1 subunit was found to restrict the relative movement of the helical and catalytic domains in the Gαi1 subunit, which is in agreement with a proposed mechanism of GDP dissociation inhibition by GoLoco motif proteins. In addition, the results provide further insights into the role of the “Switch IV” region located within the helical domain of Gα, the conformation of which might be important for interactions with various Gα partners.Electronic supplementary materialThe online version of this article (doi:10.1007/s00894-009-0516-z) contains supplementary material, which is available to authorized users.

A Point Mutation to Gαi Selectively Blocks GoLoco Motif Binding: DIRECT EVIDENCE FOR Gα·GoLoco COMPLEXES IN MITOTIC SPINDLE DYNAMICS

Heterotrimeric G-protein Galpha subunits and GoLoco motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Galphai) of Galpha subunits, and thus it is assumed that a Galphai.GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Galphai subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the alphaD-alphaE loop of the Galpha helical domain. This GoLoco-insensitivity ("GLi") mutation prevented Galphai1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Galpha subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Galphai subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gbetagamma dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Galphai subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Galphai subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Galphai.GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco motifs in receptor-mediated signal transduction.

Structural Determinants Underlying the Temperature-sensitive Nature of a Gα Mutant in Asymmetric Cell Division of Caenorhabditis elegans

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Galpha(o)) and GPA-16 (Galpha(i)) are involved in generating forces that pull on astral microtubules and position the spindle asymmetrically. GPA-16 function has been analyzed in vivo owing notably to a temperature-sensitive allele gpa-16(it143), which, at the restrictive temperature, results in spindle orientation defects in early embryos. Here we identify the structural basis of gpa-16(it143), which encodes a point mutation (G202D) in the switch II region of GPA-16. Using Galpha(i1)(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Galpha. At the permissive temperature, the mutant Galpha was stable upon GTP binding, but switch II rearrangement was compromised, as were activation state-selective interactions with regulators involved in ACD, including GoLoco motifs, RGS proteins, and RIC-8. We solved the crystal structure of the mutant Galpha bound to GDP, which indicates a unique switch II conformation as well as steric constraints that suggest activated GPA-16(it143) is destabilized relative to wild type. Spindle severing in gpa-16(it143) embryos revealed that pulling forces are symmetric and markedly diminished at the restrictive temperature. Interestingly, pulling forces are asymmetric and generally similar in magnitude to wild type at the permissive temperature despite defects in the structure of GPA-16(it143). These normal pulling forces in gpa-16(it143) embryos at the permissive temperature were attributable to GOA-1 function, underscoring a complex interplay of Galpha subunit function in ACD.

A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction

The GoLoco motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.

Gαi generates multiple Pins activation states to link cortical polarity and spindle orientation in Drosophila neuroblasts

Drosophila neuroblasts divide asymmetrically by aligning their mitotic spindle with cortical cell polarity to generate distinct sibling cell types. Neuroblasts asymmetrically localize Galphai, Pins, and Mud proteins; Pins/Galphai direct cortical polarity, whereas Mud is required for spindle orientation. It is currently unknown how Galphai-Pins-Mud binding is regulated to link cortical polarity with spindle orientation. Here, we show that Pins forms a "closed" state via intramolecular GoLoco-tetratricopeptide repeat (TPR) interactions, which regulate Mud binding. Biochemical, genetic, and live imaging experiments show that Galphai binds to the first of three Pins GoLoco motifs to recruit Pins to the apical cortex without "opening" Pins or recruiting Mud. However, Galphai and Mud bind cooperatively to the Pins GoLocos 2/3 and tetratricopeptide repeat domains, respectively, thereby restricting Pins-Mud interaction to the apical cortex and fixing spindle orientation. We conclude that Pins has multiple activity states that generate cortical polarity and link it with mitotic spindle orientation.

Structure-based Protocol for Identifying Mutations that Enhance Protein–Protein Binding Affinities

The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein–protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein–protein interface using three criteria: (1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, (2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild-type complex and (3) the mutation should not be predicted to significantly destabilize the monomers. The performance of the computational protocol was experimentally tested on two separate protein complexes; Gα i1 from the heterotrimeric G-protein system bound to the RGS14 GoLoco motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. Twelve single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. Nine of the 12 mutations successfully increased binding affinity with five of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the eight mutations identified, five were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein–protein binding affinities.

Differential G-alpha interaction capacities of the GoLoco motifs in Rap GTPase activating proteins

GoLoco motif proteins act as guanine nucleotide dissociation inhibitors (GDIs) for G-protein alpha subunits of the adenylyl cyclase-inhibitory (Gα i/o) class. Rap1GAP2 is a newly identified GoLoco motif- and RapGAP domain-containing protein, and thus is considered a potential integrator of heterotrimeric and monomeric GTPase signaling. Primary sequence analysis indicated that the Rap1GAP2 GoLoco motif contains a lysine (Lys-75), rather than an arginine, at the crucial residue responsible for binding the alpha and beta phosphates of GDP and exerting GDI activity. To determine the functional outcome of this sequence variation we conducted a biophysical analysis of the human Rap1GAP2b/c GoLoco motif. We found that human Rap1GAP2b/c was deficient in GDI activity and Gα interaction capability. Mutation of lysine-75 to arginine could not regain functional activity of the Rap1GAP2b/c GoLoco motif. Thus, the Rap1GAP2b/c GoLoco motif can be classed as inactive towards Gα subunits. We also found that the Rap1GAP1a GoLoco motif, which lacks seven N-terminal amino acid residues present in canonical GoLoco motifs, does not interact with Gα i1. In contrast, the GoLoco motif of Rap1GAP1b, which is canonical in primary sequence, was found to interact with Gα i1·GDP.

Localization of the GoLoco motif carrier regulator of G‐protein signalling 12 and 14 proteins in monkey and rat brain

Regulator of G-protein signalling (RGS)12 and -14 proteins possess the RGS domain, Ras-binding domains and the GoLoco motif. Emerging evidence suggests that these proteins are involved in several cellular functions in addition to stimulation of GTPase activity of G-protein alpha subunits. However, our understanding of the role of the two proteins in brain function remains marginal. Here, we have studied the expression pattern of RGS12 and RGS14 proteins in brain at regional, cellular and subcellular levels. Both proteins were expressed throughout the brain regions, including cortex, hippocampus, striatum, thalamus and substantia nigra. The most intense immunostaining for RGS12 was seen in cortex and that of RGS14 was found in striatum. In cortex, RGS12 and RGS14 proteins were associated with pyramidal and nonpyramidal cell types. Apical dendrites of pyramidal cells were also labelled. RGS12 was found in both nuclear and cytoplasmic compartments. In contrast to RGS12 protein, RGS14 was localized in astrocytes in addition to neurons. Pyramidal cells in the CA1 area showed labelling for both RGS proteins. The presence of RGS12 was predominantly nuclear in the striatum of rat brain; however, the labelling of this protein was non-nuclear in adult monkey brain. To our surprise, in 1-month-old monkey brain the immunostaining pattern of the same protein was changed to nuclear. Non-nuclear staining for RGS12 was also evident in thalamus of adult monkey brain; however, in 1-month-old monkey brain, it was seen into two different populations, one with nuclear and the other with cytoplasmic staining. Both RGS12 and RGS14 were exclusively localized at postsynaptic sites of excitatory synapses. Our results demonstrate a highly dynamic expression pattern of RGS12 and RGS14 proteins in the central nervous system, and support the view that these proteins may participate not only in G-protein receptor signalling pathways but also in other cellular activities.

Does WAVE1 contain a GoLoco/GPR motif?

Does WAVE1 contain a GoLoco/GPR motif?

G-protein alpha subunit interaction and guanine nucleotide dissociation inhibitor activity of the dual GoLoco motif protein PCP-2 (Purkinje cell protein-2)

Purkinje cell protein-2 (PCP-2; L7/GPSM4) is a GoLoco motif-containing protein that is specifically expressed in Purkinje and retinal ON bipolar cells. An alternative splice variant of PCP-2 has recently been isolated which contains two GoLoco motifs. Although the second GoLoco motif (GL2) of PCP-2 has been reported to interact with Gα-subunits, a complete biochemical analysis of each individual motif of PCP-2 has not been performed. We demonstrate that the first GoLoco motif (GL1) of PCP-2 is equipotent as a guanine nucleotide dissociation inhibitor (GDI) towards Gα i1 and Gα i2, while it has sevenfold lower GDI activity for Gα i3 and greater than 20-fold lower GDI activity against Gα o. In contrast we found PCP-2 GL2 to be essentially equipotent as a GDI for all Gα i subunits, but it had negligible activity toward Gα o. Using co-immunoprecipitation from COS-7 cells, we found that PCP-2 was only able to interact with Gα i1 but not Gα o nor Gα-subunits from other families (Gα s, Gα q, or Gα 12). Mutational analysis of a non-canonical residue (glycine 24) in human PCP-2 GL1 provided evidence for heterogeneity in mechanisms of Gα i interactions with GoLoco motifs. Collectively, the data demonstrate that PCP-2 is a comparatively weak GoLoco motif protein that exhibits highest affinity interactions and GDI activity toward Gα i1, Gα i2, and Gα i3 subunits.

The effect of RGS12 on PDGFβ receptor signalling to p42/p44 mitogen activated protein kinase in mammalian cells

We have previously shown that the PDGFβ receptor uses a classical GPCR-mediated pathway in order to induce efficient activation of p42/p44 MAPK in response to PDGF. We therefore, considered the possibility that GTPase accelerating proteins (RGS proteins), which regulate GPCR signalling, modulate PDGFβ receptor-mediated signal transmission. Several lines of evidence were obtained to support functional interaction between the PDGFβ receptor and RGS12 in HEK 293 and airway smooth muscle cells. Firstly, the over-expression of the RGS12 PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the PDGF-induced activation of p42/p44 MAPK. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain can form a complex with the PDGFβ receptor. Therefore, the results presented here provide the first evidence to support the concept that the PDZ/PTB domain N-terminus and/or the PTB domain of RGS12 may modulate PDGFβ receptor signalling. In airway smooth muscle cells, over-expressed recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localised with PDGFβ receptor in cytoplasmic vesicles. To provide additional evidence for a role of the PDZ/PTB domain N-terminus, we used RGS14. RGS14 has the same C-terminal domain architecture of an RGS box, tandem Ras-binding domains (RBDs) and GoLoco motif as RGS12, but lacks the PDZ/PTB domain N-terminus. In this regard, RGS14 exhibited a different sub-cellular distribution compared with RGS12, being diffusely distributed in ASM cells. These findings suggest that RGS12 via its PDZ/PTB domain N-terminus may regulate trafficking of the PDGFβ receptor in ASM cells.