Dynamics of apelin receptor/G protein coupling in living cells

During our research on apelin receptor (APJ) signalling in living cells with BRET and FRET, we demonstrated that apelin-13 stimulation can lead to the activation of Gαi₂ or Gαi₃ through undergoing a molecular rearrangement rather than dissociation in HEK293 cells expressing APJ. Furthermore, Gαo and Gαq also showed involvement in APJ activation through a classical dissociation model. However, both FRET signal and BRET ratio between fluorescent Gαi₁ subunit and Gβγ subunits demonstrated little change after apelin-13 stimulation. These results demonstrated that stimulation of APJ with apelin-13 causes activation of Gαi₂, Gαi₃, Gαo, Gαq; among which Gαi₂, Gαi₃ were activated through a novel rearrangement process. These results provide helpful data for understanding APJ mediated G-protein signalling.     

TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins

Objectives: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to G_q/11 proteins.

Methods: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP).

Results: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of G_q/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of G_iα1, β-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of G_iα1 and β-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling.

Conclusion: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.     

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors

D₂ and D₃ dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D₂ receptor couples only to inhibitory G proteins, D₃ receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D₂ and D₃ receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D₂ receptor couple to Gi protein in a pattern characteristic of D₂ receptor. On the other hand chimeras containing a third cytoplasmic loop of D₃ receptor have coupling characteristics like those of D₃ receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D₂ and D₃ to G proteins.     

Differential Effects of AGS3 Expression on D2L Dopamine Receptor-Mediated Adenylyl Cyclase Signaling

Activator of G protein signaling 3 (AGS3) binds Gα_i subunits in the GDP-bound state, implicating AGS3 as an important regulator of Gα_i-linked receptor (e.g., D2 dopamine and μ-opioid) signaling. We examined the ability of AGS3 to modulate recombinant adenylyl cyclase (AC) type 1 and 2 signaling in HEK293 cells following both acute and persistent activation of the D_2L dopamine receptor (D_2LDR). AGS3 expression modestly enhanced the potency of acute quinpirole-induced D_2LDR modulation of AC1 or AC2 activity. AGS3 also promoted desensitization of D_2LDR-mediated inhibition of AC1, whereas desensitization of D_2LDR-mediated AC2 activation was significantly attenuated. Additionally, AGS3 reduced D_2LDR-mediated sensitization of AC1 and AC2. These data suggest that AGS3 is involved in altering G protein signaling in a complex fashion that is effector-specific and dependent on the duration of receptor activation.     

Binding characteristics of a membrane receptor that recognizes 1α25-dihydroxyvitamin D3 and its epimer, 1β,25-dihydroxyvitamin D3

The Steroid hormon 1α, @5-Dihydroxyvitamin D₃ has been shown to expert rapid effect (15 s to 5 min) in osteoblast. These occur in osteoblast-like cells lacking the nuclear vitamin D receptor, ROS 24/1, suggesting that a separate signalling system mediates the rapid action. These non-genomic action include rapid activation of phospholipase C and opening of calcium channels, pointing to a membrane localization of this signalling system. Previous studies have shown that the 1β epimer of 1α25-dihydroxyvitamina D₃ can block these rapid action, indicating that the 1β epimer may bind to the recptor responsible for the rapid action sin a competative manner. We have assessed the displacement of ³H-1α,25dihydroxyvitamin D₃ by vitamin D compounds, as well as the apparent dissociation constant of 1α25-dihydroxyvitamin D₃ and its 1β epimer for the memberane receptor in membrane prepration from ROS 24/1 cells. Increasing concentrations of 1α25-dihydroxyvitamin D₃, 7.25 nM to 725 nM, displaced ³H-1α25-dihydrxyvitamin D₃ from the membranes with 725 nM of the hormone displacing 40–49% of the radioactivity. Similarly, 1β,25-dihydroxyvitamin D₃, 7.25 nM and 72.5 nM, displaced 1α25-dihydroxyvitamin D₃ binding while 25-hydroxyvitamin D₃, 7.25 nM, did not. The apparent dissociation constant (K_D) for 1α25-dihydroxyvitamin D₃ was detrermined from displacement of ³H-1α25-dihydroxyvitamin D₃ yielding a value of 8.1 × 10^?7 M by Scatchard analysis. The K_D for the 1β epimer determine from displacement of ³H-1α25-dihydroxyvitamin D₃ was 4.8 × 10^?7 M. The data suggest the presence of a receptor on the membrane of ROS 24/1 cells that reconize 1α25-dihydroxyvitamin D₃ and its 1β epimer, but not 25-dihydroxyvitamin D₃. Its ability to reconize the 1β epimer which appears to be a specific anagonist of the rapid effect of the hormone suggests that these studies may be the initial steps in the isolation and characterization of the signalling system mediating the rapid action of vitamin D.     

Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form H_II-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Gαq/11 protein and hence, signal transduction.     

Roles of GPR41 and GPR43 in leptin secretory responses of murine adipocytes to short chain fatty acids

GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Gα_i. Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Gα_i signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Gα_i signalling mediated by GPR43 in SCFA-stimulated leptin secretion.     

Lipid rafts as platforms for sphingosine 1-phosphate metabolism and signalling

Spontaneous segregation of cholesterol and sphingolipids as a liquid-ordered phase leads to their clustering in selected membrane areas, the lipid rafts. These specialized membrane domains enriched in gangliosides, sphingomyelin, cholesterol and selected proteins involved in signal transduction, organize and determine the function of multiprotein complexes involved in several aspects of signal transduction, thus regulating cell homeostasis. Sphingosine 1-phosphate, an important biologically active mediator, is involved in several signal transduction processes regulating a plethora of cell functions and, not only several of its downstream effectors tend to localize in lipid rafts, some of the enzymes involved in its pathway, of receptors involved in its signalling and its transporters have been often found in these membrane microdomains. Considering this, in this review we address what is currently known regarding the relationship between sphingosine 1-phosphate metabolism and signalling and plasma membrane lipid rafts.     

Dynamics of membrane lipid domains in neuronal cells differentiated in culture

Treatment with methyl-beta-cyclodextrin (MCD) induced a time- and dose-dependent efflux of cholesterol, sphingolipids, and phosphatidylcholine (PC) from cerebellar neurons differentiated in culture. With a "mild" treatment, the loss of cell lipids induced a deep reorganization of the remaining membrane lipids. In fact, the amount of PC associated with a Triton X-100-insoluble membrane fraction (highly enriched in sphingolipids and cholesterol in nontreated cells) was lowered by the treatment. This suggested a reduction of the lipid domain area. However, the cholesterol and sphingolipid enrichment of this fraction remained substantially unchanged, suggesting the existence of dynamic processes aimed at preserving the segregation of cholesterol and sphingolipids in membrane domains. Under these conditions, the lipid membrane domains retained the ability to sort signaling proteins, such as Lyn and c-Src, but cells displayed deep alterations in their membrane permeability. However, normal membrane permeability was restored by loading cells with cholesterol. When MCD treatment was more stringent, a large loss of cell lipids occurred, and the lipid domains were much less enriched in cholesterol and lost the ability to sort specific proteins. The loss of the integrity and properties of lipid domains was accompanied by severe changes in the membrane permeability, distress, and eventually cell death.     

Molecular organization and dynamics of the melatonin MT1 receptor/RGS20/Gi protein complex reveal asymmetry of receptor dimers for RGS and Gi coupling

Functional asymmetry of G‐protein‐coupled receptor (GPCR) dimers has been reported for an increasing number of cases, but the molecular architecture of signalling units associated to these dimers remains unclear. Here, we characterized the molecular complex of the melatonin MT₁ receptor, which directly and constitutively couples to G_i proteins and the regulator of G‐protein signalling (RGS) 20. The molecular organization of the ternary MT₁/G_i/RGS20 complex was monitored in its basal and activated state by bioluminescence resonance energy transfer between probes inserted at multiple sites of the complex. On the basis of the reported crystal structures of G_i and the RGS domain, we propose a model wherein one G_i and one RGS20 protein bind to separate protomers of MT₁ dimers in a pre‐associated complex that rearranges upon agonist activation. This model was further validated with MT₁/MT₂ heterodimers. Collectively, our data extend the concept of asymmetry within GPCR dimers, reinforce the notion of receptor specificity for RGS proteins and highlight the advantage of GPCRs organized as dimers in which each protomer fulfils its specific task by binding to different GPCR‐interacting proteins.     

Sterol structure dependence of insulin receptor and insulin-like growth factor 1 receptor activation

The plasma membrane is a dynamic environment with a complex composition of lipids, proteins, and cholesterol. Areas enriched in cholesterol and sphingolipids are believed to form lipid rafts, domains of highly ordered lipids. The unique physical properties of these domains have been proposed to influence many cellular processes. Here, we demonstrate that the activation of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) depends critically on the structures of membrane sterols. IR and IGF1R autophosphorylation in vivo was inhibited by cholesterol depletion, and autophosphorylation was restored by the replacement with exogenous cholesterol. We next screened a variety of sterols for effects on IR activation. The ability of sterols to support IR autophosphorylation was strongly correlated to the propensity of the sterols to form ordered domains. IR autophosphorylation was fully restored by the incorporation of ergosterol, dihydrocholesterol, 7-dehydrocholesterol, lathosterol, desmosterol, and allocholesterol, partially restored by epicholesterol, and not restored by lanosterol, coprostanol, and 4-cholesten-3-one. These data support the hypothesis that the ability to form ordered domains is sufficient for a sterol to support ligand-induced activation of IR and IGF1R in intact mammalian cells.     

Development of inhibitors of heterotrimeric Gαi subunits

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gα_i by competing for the GPCR and Gβγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α₂-adrenoceptor (α₂AR) mediated decreases in cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for Gα_i. Molecular docking was used to identify potential molecules that bind to and stabilize the Gα_i–GDP complex by directly interacting with both Gα_i and GDP. Gα_i–GTP and Gα_q–GDP were used as a computational counter screen and Gα_q–GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gα_i. Several compounds showed Gα_i specific inhibition and were able to block α₂AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.     

Detergent-free solubilisation & purification of a G protein coupled receptor using a polymethacrylate polymer

G protein coupled receptors (GPCRs) function as guanine nucleotide exchange factors (GEFs) at heterotrimeric G proteins, and conduct this role embedded in a lipid bilayer. Detergents are widely used to solubilise GPCRs for structural and biophysical analysis, but are poor mimics of the lipid bilayer and may be deleterious to protein function. Amphipathic polymers have emerged as promising alternatives to detergents, which maintain a lipid environment around a membrane protein during purification. Of these polymers, the polymethacrylate (PMA) polymers have potential advantages over the most popular styrene maleic acid (SMA) polymer, but to date have not been applied to purification of membrane proteins. Here we use a class A GPCR, neurotensin receptor 1 (NTSR1), to explore detergent-free purification using PMA. By using an NTSR1-eGFP fusion protein expressed in Sf9 cells, a range of solubilisation conditions were screened, demonstrating the importance of solubilisation temperature, pH, NaCl concentration and the relative amounts of polymer and membrane sample. PMA-solubilised NTSR1 displayed compatibility with standard purification protocols and millimolar divalent cation concentrations. Moreover, the receptor in PMA discs showed stimulation of both G_q and G_i1 heterotrimers to an extent that was greater than that for the detergent-solubilised receptor. PMA therefore represents a viable alternative to SMA for membrane protein purification and has a potentially broad utility in studying GPCRs and other membrane proteins.     

Receptor-Mediated Activation of G Proteins Is Increased in Postmortem Brains of Bipolar Affective Disorder Subjects

Abstract: Guanine nucleotide binding proteins (G proteins) have been implicated in the pathophysiology of bipolar affective disorder. In the present investigation receptor-mediated G protein activation and changes in G protein trimeric state were examined in frontal cortical membranes obtained from postmortem brains of bipolar affective disorder subjects and from age-, sex-, and postmortem interval-matched controls. Stimulation of cortical membranes with serotonin, isoproterenol, or carbachol increased guanosine 5′-O-(3-[³⁵S]thiophosphate) ([³⁵S]GTPγS) binding to specific G_α proteins in a receptor-selective manner. The abilities of these receptor agonists to stimulate the binding of [³⁵S]GTPγS to the G_α proteins was enhanced in membranes from bipolar brains. Immunoblot analyses showed increases in the levels of membrane 45- and 52-kDa G_αs proteins but no changes in the amounts of G_αi, G_αo, G_αz, G_αq/11, or G_β proteins in membrane or cytosol fractions of bipolar brain homogenates. Pertussis toxin (PTX)-activated ADP-ribosylations of G_αi and G_αo were enhanced by ～80% in membranes from bipolar compared with control brains, suggesting an increase in the levels of the trimeric state of these G proteins in bipolar disorder. Serotonin-induced, magnesium-dependent reduction in PTX-mediated ADP-ribosylation of G_αi/G_αo in cortical membranes from bipolar brains was greater than that observed in controls, providing further evidence for enhanced receptor-G protein coupling in bipolar brain membranes. In addition, the amounts of G_β proteins that coimmunoprecipitated with the G_α proteins were also elevated in bipolar brains. The data show that in bipolar brain membrane there is enhanced receptor-G protein coupling and an increase in the trimeric state of the G proteins. These changes may contribute to produce exaggerated transmembrane signaling and to the alterations in affect that characterize bipolar affective disorder.     

The γ-aminobutyric acid receptor B, but not the metabotropic glutamate receptor type-1, associates with lipid rafts in the rat cerebellum

Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non-ionic detergent-extraction at 4 degrees C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic gamma-aminobutyric acid receptor B (GABA(B) receptor) and the metabotropic glutamate receptor-1 from rat cerebellum are insoluble in the non-ionic detergent Triton X-100. However, only the GABA(B) receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABA(B) receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl-beta-cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABA(B) receptor. These properties are all consistent with an association of the GABA(B) receptor with lipid raft microdomains.     

Human Substance P Receptor Expressed in Sf9 Cells Couples with Multiple Endogenous G Proteins

Abstract

To identify the G proteins involved in the function of human substance P receptor (hSPR), the receptor was expressed in Sf9 cells using the baculovirus expression system. Maximal hSPR expression was up to 65 pmol/mg membrane protein. The following data indicated that hSPR in Sf9 membranes is coupled to endogenous G proteins: 1) binding of agonist radioligand [¹²⁵I]BHSP to the receptor was sensitive to guanine nucleotides; and 2) stimulation of the receptor increased [³⁵S]GTPγS binding. The hSPR-associated G proteins were identified by photoaffinity labeling with [α-³²P]-azidoanilido GTP ([α-³²P]AAGTP), followed by immunoprecipitation of the labeled G proteins with antibodies specific for various Gα-subunits. These experiments showed that stimulation of hSPR in Sf9 membranes activated multiple endogenous G proteins including Gα_o, Gα^q/11, and Gα. While hSPR's ability to associate with G^q/11 is well-documented, the present study provides the first evidence of hSPR's potential to activate Gα_o and Gα_s.     

Endogenous RGS proteins facilitate dopamine D2S receptor coupling to Gαo proteins and Ca2+ responses in CHO-K1 cells

The role of RGS proteins on dopaminergic D_2S receptor (D_2SR) signalling was investigated in Chinese hamster ovary (CHO)-K1 cells, using recombinant RGS protein- and PTX-insensitive G_αo proteins. Dopamine-mediated [³⁵S]GTPγS binding was attenuated by more than 60% in CHO-K1 D_2SR cells coexpressing a RGS protein- and PTX-insensitive G_αoGly¹⁸⁴Ser:Cys³⁵¹Ile protein versus cells coexpressing a similar amount of PTX-insensitive G_αoCys³⁵¹Ile protein. Dopamine-agonist-mediated Ca²⁺ responses were dependent on the coexpression with a G_αoCys³⁵¹Ile protein and were fully abolished upon coexpression with a G_αoGly¹⁸⁴Ser:Cys³⁵¹Ile protein. These results suggest that interactions between the G_αo protein and RGS proteins are involved in efficient D_2SR signalling.     

Oiling the key hole

Many bacteria have been found to interact with specialized domains, rich in cholesterol and sphingolipids, of the host plasma membrane, termed lipid rafts. The mechanisms that underlie this interaction are starting to be unravelled. In this issue, Hayward et al. show that early effector proteins secreted by type III secretion harbouring Gram-negative bacteria are in fact cholesterol-binding proteins. Combined with other recent findings, this work shows that multiple steps leading to infection by these bacteria depend on raft components: activation of secretion, binding, perforation of the host cell membrane and signalling to trigger bacterial engulfment.     

Physicochemical Modulation of Agonist-Induced [35S]GTPγS Binding: Implications for Coexistence of Multiple Functional Conformations of Dopamine D1-Like Receptors

Dopamine agonist-stimulated [³⁵S]GTPγS binding to membrane G proteins was studied in select brain regions under experimental conditions that permit the activation of receptor coupling to the G proteins G_i, G_s, or G_q. Agents studied were agonists known to be effective at various dopamine receptor/effector systems and included quinelorane (D₂-like/G_i), SKF38393 (D₁-like/G_q, D₁-like/G_s), SKF85174 (D₁-like/G_s), and SKF83959 (D₁-like/G_q). Dopamine and SKF38393 significantly stimulated [³⁵S]GTPγS binding to normal striatal membranes by 161% and 67% above controls. Deoxycholate, which enhances agonist-induced phospholipase C (PLC) stimulation, markedly enhanced the agonistic effects of dopamine and SKF38393 to 530% and 637% above controls, respectively. The enhancing effects of deoxycholate were reversed if it was washed off the membranes before agonist addition. The thiol-reducing agent, dithiothreitol, completely abolished the effects of SKF38393 and SKF83959, whereas SKF85174 effects were augmented. Agonist responses were concentration-related, and highest efficacies were obtained in the hippocampus, thus paralleling both the brain regional distribution and agonist efficacies previously observed in phosphoinositide hydrolysis assays. These findings suggest that D₁-like receptor conformations that mediate agonist stimulation of G_s/adenylylcyclase may be structurally different from those that mediate G_q/PLC activation. Although the exact mechanism of deoxycholate's effect awaits elucidation, the results are consistent with the emerging concept of functional selectivity whereby deoxycholate could create a membrane environment that facilitates the transformation of the receptor from a conformation that activates G_s/adenylylcyclase to one that favors G_q/PLC signaling.     

Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane

Aquaporin-1 (AQP1) is an integral membrane protein that facilitates osmotic water transport across cell plasma membranes in epithelia and endothelia. AQP1 has no known specific interactions with cytoplasmic or membrane proteins, but its recovery in a detergent-insoluble membrane fraction has suggested possible raft association. We tracked the membrane diffusion of AQP1 molecules labeled with quantum dots at an engineered external epitope at frame rates up to 91 Hz and over times up to 6 min. In transfected COS-7 cells, >75% of AQP1 molecules diffused freely over ∼7 μm in 5 min, with diffusion coefficient, D_1-3 ∼ 9 × 10⁻¹⁰ cm²/s. In MDCK cells, ∼60% of AQP1 diffused freely, with D_1-3 ∼ 3 × 10⁻¹⁰ cm²/s. The determinants of AQP1 diffusion were investigated by measurements of AQP1 diffusion following skeletal disruption (latrunculin B), lipid/raft perturbations (cyclodextrin and sphingomyelinase), and bleb formation. We found that cytoskeletal disruption had no effect on AQP1 diffusion in the plasma membrane, but that diffusion was increased greater than fourfold in protein de-enriched blebs. Cholesterol depletion in MDCK cells greatly restricted AQP1 diffusion, consistent with the formation of a network of solid-like barriers in the membrane. These results establish the nature and determinants of AQP1 diffusion in cell plasma membranes and demonstrate long-range nonanomalous diffusion of AQP1, challenging the prevailing view of universally anomalous diffusion of integral membrane proteins, and providing evidence against the accumulation of AQP1 in lipid rafts.