The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

A critical evaluation of choice negotiation for patient-centred medicine and psychotherapy.

STUDY AIMS: The present paper aimed at discussing how the process of decision-making should be taken care of in healthcare services. METHODS: This is a position paper based on a review of the relevant literature about meaning-making processes in medical encounters and psychotherapy. DISCUSSION: Authors argued that choice options could be perceived as meaningful by patients if their uncertainties were taken into account and grounded on mutual understanding and reciprocal trust. To this end, any decision-making process should satisfy the patient's legitimate expectations by making choices and habits compatible. CONCLUSION: In depht analysis of meaning-making processes is crucial for better refining good practices of shared decision-making.

Thick description and systems thinking: Reiterating the importance of a biopsychosocial approach to mental health.

STUDY AIMS: The article aims at reiterating the importance of a biopsychosocial approach to mental health, taking stock of the critiques that have been raised and moving forward throughout a reconsideration of the theoretical background of systems thinking and emphasizing the relevance of the concept of thick description for the promotion of an adequate reflection on methodology and case formulation. LITERATURE REVIEW: It is our opinion that the biopsychosocial approach is still a powerful framework for making sense of the growing data collected in the different fields related to mental health and for designing proper treatment plans. A crucial challenge for mental health is that of surpassing the dichotomies and ideological disputes that still contaminate the field with detrimental effects on the advancement of knowledge and on the integration and continuity of different kind of interventions. CONCLUSIONS: The time is ripe for building bridges among neuroscience, humanities and social sciences, and this can only happen within the umbrella of a biopsychosocial perspective reinstated into its systems thinking background.

Characterization of Collagen/Lipid Nanoparticle-Curcumin Cryostructurates for Wound Healing Applications.

Curcumin-loaded collagen cryostructurates have been devised for wound healing applications. Curcumin displays strong antioxidant, antiseptic, and anti-inflammatory properties, while collagen is acknowledged for promoting cell adhesion, migration and differentiation. However, when curcumin is loaded directly into collagen hydrogels, it forms large molecular aggregates and clogs the matrix pores. A double-encapsulation strategy is therefore developed by loading curcumin into lipid nanoparticles (LNP), and embedding these particles inside collagen scaffolds. The resulting collagen/LNP cryostructurates have an optimal fibrous structure with ≈100 µm average pore size for sustaining cell migration. Results show that collagen is structurally unaltered and that nanoparticles are homogeneously distributed amidst collagen fibers. Hydrogels soaked in saline buffer release about 20 to 30% of their nanoparticles content within 24 h, while achieved 100% release after 25 days. When exposed to NIH 3T3 fibroblasts, these hydrogels provide a satisfactory scaffold for cell interaction as early as 4 h after seeding, with no cytotoxic counter effect. These positive features make the collagen/lipid cryostructurates a promising material for further use in wound healing.

Dichlorodiphenyltrichloroethane (DDT) induced extracellular vesicle formation: a potential role in organochlorine increased risk of Parkinson's disease.

A number of studies have demonstrated that rural living and exposure to pesticides such as dichlorodiphenyltrichloroethane (DDT) highly increase the chances of developing Parkinson's disease. In a previous work, we have found that DDT leads to the formation of vesicular buds that are released from the cells upon fusion of an intermediate endocytic compartment with the plasma membrane. Since extracellular vesicles like exosomes have been implicated in the development of neurodegenerative diseases through the propagation of neurotoxic misfolded proteins from neuron to neuron, in this minireview we propose that organochlorine pesticides could enhance the risk of neurodegenerative diseases by increasing the formation of exosomes.

Ultrastructural investigation on fibroblast interaction with collagen scaffold.

Collagen-based scaffolds are used as temporary or permanent coverings to help wound healing. Under natural conditions, wound healing is affected by such factors as cell types, growth factors and several components of the extracellular matrix. Due to the complexity of the cell-to-matrix interaction, many cell based mechanisms regulating wound healing in vivo are not yet properly understood. However, the whole process can be partially simulated in vitro to determine how cells interact with the collagen scaffold in relation to such features as physico-chemical properties, matrix architecture and fiber stability. Under these conditions, cell migration into the collagen matrix can be easily assessed and causally correlated with these features. In this study, we aimed at providing a structural analysis of how NIH3T3 fibroblasts migrate and proliferate in vitro when seeded on a native type-I collagen scaffold. To this end, samples were collected at regular time intervals and analyzed by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Through this experimental approach we demonstrate that collagen is gradually frayed into progressively thinner fibrils as fibroblasts migrate into the matrix, embrace the collagen fibers with long filopodia and form large intracellular vacuoles. A key role in this process is also played by microvesicles shed from the fibroblast plasma membrane and spread over long distances inside the collagen matrix. These observations indicate that a native type-I equine collagen provides favorable conditions for simulating collagen processing in vitro and eventually for unraveling the mechanisms controlling cell uptake and intracellular degradation.

Microvesicles shed from fibroblasts act as metalloproteinase carriers in a 3-D collagen matrix.

This study shows that fibroblasts migrating into a collagen matrix release numerous microvesicles into the surrounding medium. By spreading in regions of the matrix far distant from cells of origin, microvesicles carry metalloproteinase 9 (MMP-9) to act upon the collagen fibrils. As a result, the collagen matrix is gradually transformed from a laminar to a fibrillar type of architecture. As shown by western blots and gelatin zymography, MMP-9 is secreted as a 92 kDa precursor and activated upon release of 82 kDa product into the culture medium. Activation is more efficient under three-dimensional than in two-dimensional culturing conditions. While MMP-9 labeling is associated with intraluminal vesicles clustered inside the microvesicles, the microvesicle's integrin ß1 marker is bound to the outer membrane. The intraluminal vesicles are recruited from the cortical cytoplasm and eventually released following uploading inside the microvesicle. Here, we propose that fusion of the intraluminal vesicles with the outer microvesicle's membrane could work as a mechanism controlling the extent to which MMP-9 is first activated and then released extracellularly.

Towards a heterarchical approach to biology and cognition.

In this article we challenge the pervasive notion of hierarchy in biological and cognitive systems and delineate the basis for a complementary heterarchical approach starting from the seminal ideas of Warren McCullock and Gregory Bateson. We intend these considerations as a contribution to the different scientific disciplines working towards a multilevel integrative perspective of biological and cognitive processes, such as systems and integrative biology and neuroscience, social and cultural neuroscience, social signal transduction and psychoneuroimmunology, for instance. We argue that structures and substrates are by necessity organized hierarchically, while communication processes - and their embeddedness - are rather organized heterarchically. Before getting into the implications of the heterarchical approach and its congeniality with the semiotic perspective to biology and cognition, we introduce a set of notions and concepts in order to advance a framework that considers the heterarchical embeddedness of different layers of physiological, behavioral, affective, cognitive, technological and socio-cultural levels implicit in networks of interacting minds, considering the dynamic complementarity of bottom-up and top-down causal links. This should contribute to account for the integration, interpretation and response to complex aggregates of information at different levels of organization in a developmental context. We illustrate the dialectical nature of embedded heterarchical processes by addressing the simultaneity and circularity of cognition and volition, and how such dialectics can be present in primitive instances of proto-cognition and proto-volition, giving rise to our claim that subjectivity and semiotic freedom are scalar properties. We collate the framework with recent empirical systemic approaches to biology and integrative neuroscience, and conclude with a reflection on its implications to the understanding of the emergence of pathological conditions in multi-level semiotic systems.

Severe and polytraumatic injuries among recreational skiers and snowboarders: incidence, demographics and injury patterns in South Tyrol.

BACKGROUND: Alpine skiing and snowboarding are popular winter sports. The practice of these sports is related to traumatic injuries, some of which are severe and/or life threatening. OBJECTIVES: To identify the incidence, injury patterns and associated risk factors of severe and polytraumatic injuries in South Tyrol. MATERIALS AND METHODS: During four consecutive winter seasons (2001-2005), data of every patient referred to our emergency department (Bolzano-Bozen) after a skiing or snowboarding accident were collected. One hundred and five patients with an Injury Severity Score of 16 or higher were identified (90 skiers, 15 snowboarders). Statistical descriptive analyses were carried out by producing frequency tables. Chi-square test was performed to verify possible association between injury severity and type of sport. Risk factors for severe injuries were evaluated using logistic regression with robust variance estimators. RESULTS: Traumatic brain injury was the most common injury observed (51 cases), followed by vertebral injury (45 cases); 63% of the patients reported two or more associated injuries. We observed significant associations between severe spine injuries and the following risk factors: snowboarders who reported more severe injuries than skiers [odds ratio=5.89, 95% confidence interval (CI)=1.31, 26.44], age classes of 40-50 years and over 60 years showed an OR of 8.10 (95% CI=1.87, 35.06) and 5.16 (95% CI=1.27, 21.01), respectively, with respect to age class (20-40 years). CONCLUSION: Severe traumatic injuries occur among skiers and snowboarders, and preventive measures such as the use of helmets and educational programs, are necessary.

The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) alters the membrane raft location of the TSH receptor stably expressed in Chinese hamster ovary cells.

DDT is a highly lipophilic molecule known to deplete membrane rafts of their phosphoglycolipid and cholesterol contents. However, we have recently shown that DDT can also alter the thyroid homeostasis by inhibiting TSH receptor (TSHr) internalization. The present study was undertaken to verify whether DDT goitrogenic effects are due to the insecticide acting directly on TSHr or via alteration of the membrane rafts hosting the receptor itself. Our results demonstrate that, in CHO-TSHr transfected cells, TSHr is activated in the presence of TSH, while it is inhibited following DDT exposure. DDT can also reduce the endocytic vesicular traffic, alter the extension of multi-branched microvilli along their plasma membranes and induce TSHr shedding in vesicular forms. To verify whether TSHr displacement might depend on DDT altering the raft constitution of CHO-TSHr cell membranes the extent of TSHr and lipid raft co-localization was examined by confocal microscopy. Evidence shows that receptor/raft co-localization increased significantly upon exposure to TSH, while receptors and lipid rafts become dislodged on opposite cell poles in DDT-exposed CHO-TSHr cells. As a control, under similar culturing conditions, diphenylethylene, which is known to be a lipophilic substance that is structurally related to DDT, did not affect the extent of TSHr and lipid raft co-localization in CHO-TSHr cells treated with TSH. These findings corroborate and extend our view that, in CHO cells, the DDT disrupting action on TSHr is primarily due to the insecticide acting on membranes to deplete their raft cholesterol content, and that the resulting inhibition on TSHr internalization is due to receptor dislodgement from altered raft microdomains of the plasma membrane.

A morphological and biochemical analysis comparative study of the collagen products Biopad, Promogram, Puracol, and Colactive.

The aim of this study was to compare the capacity of the collagen products Biopad (Euroresearch, Milano, Italy), Promogran (Systagenix Wound Management, Quincy, Massachusetts), Colactive (Smith & Nephew, St Petersburg, Florida), and Puracol (Medline Industries, Mundelein, Illinois) to interact with biological tissues and to start restoring the healing process. These results demonstrate how these products can interact differently with enzymes and cells that characterize the environment of a healing wound.

Factors affecting injury severity among recreational skiers and snowboarders: an epidemiology study.

Different results have been reported for skiing and snowboarding injuries worldwide. Few studies consider the injury severity score (ISS) for the evaluation of differences among injured skiers-snowboarders. The aim of this study is to identify possible risk factors that affect the severity of skiing and snowboarding injuries in three winter seasons (2002-2005) in South Tyrol. For every injured skier or snowboarder referred to our emergency department in three consecutive seasons, the following data were collected: date of birth, gender, self-declared technical skills level, place of residence (local/non-local), as well as the date, time, and place of the accident. Type of injury and ISS were retrospectively assigned. Data concerning the snowfall in the last 24 h, average snow level, and outdoor air temperature values were obtained from four weather stations that were located inside the ski resorts. A multiple linear regression model was used to evaluate the association between ISS and potential determinants. In the analyzed seasons, 2,511 injured skiers and 843 injured snowboarders were evaluated at our emergency department. There was a significant change in the ISS value for subjects with different self-reported skills levels (P < 0.001). Men and non-local residents experienced more severe injuries than women and local residents, respectively (P < 0.013, P < 0.001). The ISS was higher for people aged over 60 (P < 0.001). Snowfalls brought about a decrease in accident severity (P = 0.009). The severity of the injuries increases with age. Prevention and information programs should be targeted to people who are at high risk of severe injury. A 24-h fresh snowfall seems to reduce the severity of injuries. Very little is known about snow conditions and winter sports injury. Further studies are needed to explore this field.

Presence of a putative steroidal allosteric site on glycoprotein hormone receptors.

In a previous work we found that the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), inhibits the accumulation of cAMP as induced by the bovine thyroid stimulating hormone (bTSH) in cells transfected with the TSH receptor. In this work, we demonstrate that the DDT molecular analogues, diethylstilbestrol and quercetine, are more potent inhibitors of the TSH receptor activity than DDT itself. The notion that all these compounds interfere with nuclear estrogen receptors, as either agonists (DDT and diethylstilbestrol) or antagonists (quercetin), prompted us to test the ability of the steroid hormone 17-beta-estradiol to inhibit the TSH receptor activity. We found that estrogen exposure causes a modest but significant inhibition of the bTSH induced cAMP accumulation both in transfected CHO-TSH receptor and Fischer Rat Thyroid Low Serum 5% (FRTL-5) cells. When applied to CHO cells transfected with the luteinizing hormone receptor, 17-beta-estradiol proved capable of inhibiting the hCG induced cAMP accumulation at a concentration as low as 10nM, though the effect was not greater than 35%. The effect of 17-beta-estradiol was not estrogen receptors mediated, as co-transfection of the estrogen receptor alpha and beta subunits with LH receptor caused cAMP to increase above the level attained by the sole hCG stimulation, and not to decrease it as expected. These data suggest the presence of a steroidal-like allosteric binding site on glycoprotein hormone receptors.

Sortilin is a putative postendocytic receptor of thyroglobulin.

The Vps10p family member sortilin is involved in various cell processes, including protein trafficking. Here we found that sortilin is expressed in thyroid epithelial cells (thyrocytes) in a TSH-dependent manner, that the hormone precursor thyroglobulin (Tg) is a high-affinity sortilin ligand, and that binding to sortilin occurs after Tg endocytosis, resulting in Tg recycling. Sortilin was found to be expressed intracellularly in thyrocytes, as observed in mouse, human, and rat thyroid as well as in FRTL-5 cells. Sortilin expression was demonstrated to be TSH dependent, both in FRTL-5 cells and in mice treated with methimazole and perchlorate. Plasmon resonance binding assays showed that Tg binds to sortilin in a concentration-dependent manner and with high affinity, with Kd values that paralleled the hormone content of Tg. In addition, we found that Tg and sortilin interact in vivo and in cultured cells, as observed by immunoprecipitation, in mouse thyroid extracts and in COS-7 cells transiently cotransfected with sortilin and Tg. After incubation of FRTL-5 cells with exogenous, labeled Tg, sortilin and Tg interacted intracellularly, presumably within the endocytic pathway, as observed by immunofluorescence and immunoelectron microscopy, the latter technique showing some degree of Tg recycling. This was confirmed in FRTL-5 cells in which Tg recycling was reduced by silencing of the sortilin gene and in CHO cells transfected with sortilin in which recycling was increased. Our findings provide a novel pathway of Tg trafficking and a novel function of sortilin in the thyroid gland, the functional impact of which remains to be established.

The thyroid disruptor 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane appears to be an uncompetitive inverse agonist for the thyrotropin receptor.

In this study, we aimed at establishing whether two previously identified thyroid disruptors, the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and Aroclor 1254 (a complex mixture of polychlorinated water), may inhibit thyrotropin (TSH) receptor (TSHr) activity. DDT and Aroclor 1254 were shown to inhibit both the basal and bovine TSH (bTSH)-stimulated accumulation of cAMP in Chinese hamster ovary (CHO)-K1 cells stably transfected with the TSHr. Furthermore, both DDT and Aroclor 1254 did indeed prevent cAMP accumulation, as induced by the constitutive activity of a point mutant TSHr(I486M) transiently transfected in African green monkey kidney fibroblast (COS)-7 cells. Neither trypsin digestion of the extracellular domain (ECD) nor deletion of the ECD in a mutant TSHr trunk transiently transfected in COS-7 cells counteracted the inhibitory activity of DDT and Aroclor 1254. DDT exerted a weak inhibitory activity against forskolin in both CHO-K1 and COS-7 cells, whereas it was nil against the agonists dopamine and 5'-(N-ethyl-carboxamido)-adenosine (NECA) in CHO cells stably transfected with the dopamine D1 receptor and in COS-7 cells transiently transfected with the adenosine type 2a receptor (A2a) receptor. Furthermore, DDT was inactive against the stimulation by isoproterenol of the endogenously expressed beta2 adrenergic receptor in COS-7 cells. Conversely, Aroclor 1254 inhibited completely forskolin activity in CHO-K1 cells but not in COS-7 cells. Furthermore, it did not prevent accumulation of cAMP as induced by NECA in A2a transfected cells. The analog of DDT, diphenylethylene, was inactive against bTSH-induced increase in cAMP in CHO-K1 cells stably transfected with the TSHr. We interpreted these results as indicating that DDT and possibly Aroclor 1254 may have an uncompetitive inverse agonist activity for the TSHr.

Sledding injuries: is safety in this winter pastime overlooked? A three-year survey in South-Tyrol.

BACKGROUND: Sledding is a popular traditional pastime in northern countries. However it is only rarely thought as a potentially dangerous activity even though serious injuries and deaths do occur. The purpose of this study was to calculate the incidence, the severity and the pattern of sledding-related injuries in our area, in order to set up possible preventive measures. RESULTS: In three consecutive winter seasons (Dec.-Apr.,2002-2005). 356 patients (182 males, 174 females, mean age 26.9 years, range 2 to 81) were referred directly to our ED after a sledding injury. One patient (male, age 21 years) was transferred from a community hospital and died on the following day. Two patients (males, age 47 and 28 years) were declared dead on the scene. In the majority of the cases the accident was due to a fall and collision with the ground or a standing object. The number of injuries showed a progressive increase during the observed seasons and all deadly accidents were observed in the last season. Injuries were divided into three severity classes: minor (ISS /= 4 < 15), severe (ISS >/= 15). Minor and intermediate injuries were equally distributed between males and females, whereas all severe and deadly accidents occurred to male patients. Time of accident and place of accident did not affect the injury severity. A total of 386 lesions were detected. The most common diagnosis was head trauma (14,5%), followed by knee sprain (13%), ankle sprain (11.5%), and ankle/leg fracture (9%). 41 patients required hospital admission. The mean hospital length of stay was 3.9 days and 16 patients required surgery. The most common diagnosis on admission was lower limb fracture (13 patients) and head trauma (13 patients). The percentage of pediatric injuries was much lower than that reported in other studies. CONCLUSION: Sledding is rarely thought of as a potentially dangerous activity, but it can result in serious injury. Better public awareness of the risks of sledding injuries is required and preventive measures like the use of helmet, soft-side protections on the tracks, regular checks of the track conditions and good lightning for night sledding should be enforced.

Resting smooth muscle cells as a model for studying vascular cell activation.

Vascular smooth muscle (VSM) cells constitute the main structural components of tunica media. Under physiological conditions, these cells display a contractile phenotype and a low proliferative activity. However, they may also acquire a synthetic phenotype and become predominantly proliferative if stimulated under certain stress conditions. This capacity plays a major role in the inception and progression of such cardiovascular diseases as atherosclerosis, hypertension and restenosis. Porcine coronary smooth muscle (PCSM) cells exhibit a synthetic phenotype (ON cells) under standard culturing conditions, but they can be reverted to a contractile phenotype (OFF cells) in a serum-free medium. However, OFF cells can also re-acquire a synthetic phenotype (OFF/ON cells) upon serum administration. In the present study, proliferative and contractile behaviors were characterized by expression of specific differentiation markers. Taken together, these results demonstrate that porcine vascular smooth muscle cells can retain their phenotypic plasticity in culture, and thus mimic in vitro their in vivo differentiation states. OFF cells may thus provide a suitable model system in studying the mechanism(s) by which either known or unknown serum factors may trigger vascular smooth muscle activation. In the present study, this possibility was actually tested by exposing OFF cells to fetal bovine serum (FBS), PDGF-BB and IGF-I. Data show that only FBS could induce a synthetic phenotype in OFF cells, while both PDGF-BB and IGF-I failed to induce any VSM activation.

Ablation of non functioning renal allograft by embolization: a valid alternative to graft nephrectomy?

OBJECTIVE: Removal of transplant for the treatment of graft intolerance syndrome (GIS) is an invasive procedure with high risk, often performed in patients with poor general conditions. Renal allograft embolization is a recent alternative treatment to surgical nephrectomy. The aim of this study was to evaluate the efficacy and safety of allograft embolization in a series of patients with GIS. PATIENTS AND METHODS: The study included 12 patients (9 males and 3 females) with irreversible renal graft rejection and GIS. All patients were in hemodialysis and they have not responded to medical treatment. Infection was ruled out by blood and urine cultures. The embolization was performed using polyvinyl alcohol particles and steel coils. Vascular access was obtained via femoral artery puncture in all the patients. Before starting embolization at the puncture site local anaesthesia was performed. RESULTS: Eleven of the twelve procedures were technically successful, but in one patient a second treatment was necessary, after a month, for the presence of collateral perirenal circulation caused hematuria. There were no major complications and the mean hospital stay was 5 days. CONCLUSION: The graft embolization is a simple, safe and effective technique that permits non-surgical ablation of a non-functioning renal allograft in a significant number of patients.

Paired activation of two components within muscarinic M3 receptor dimers is required for recruitment of beta-arrestin-1 to the plasma membrane.

beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.