Distinct role of long 3' UTR BDNF mRNA in spine morphology and synaptic plasticity in hippocampal neurons.

The brain produces two brain-derived neurotrophic factor (BDNF) transcripts, with either short or long 3' untranslated regions (3' UTRs). The physiological significance of the two forms of mRNAs encoding the same protein is unknown. Here, we show that the short and long 3' UTR BDNF mRNAs are involved in different cellular functions. The short 3' UTR mRNAs are restricted to somata, whereas the long 3' UTR mRNAs are also localized in dendrites. In a mouse mutant where the long 3' UTR is truncated, dendritic targeting of BDNF mRNAs is impaired. There is little BDNF in hippocampal dendrites despite normal levels of total BDNF protein. This mutant exhibits deficits in pruning and enlargement of dendritic spines, as well as selective impairment in long-term potentiation in dendrites, but not somata, of hippocampal neurons. These results provide insights into local and dendritic actions of BDNF and reveal a mechanism for differential regulation of subcellular functions of proteins.

The Effect of Helium Ion Radiation on the Material Properties of Bone.

Ionizing radiation, from both space and radiation therapy, is known to affect bone health. While there have been studies investigating changes in bone density and microstructure from radiation exposure, the effects of radiation on material properties are unknown. The current study addresses this gap by assessing bone material property changes in rats exposed to helium-4 radiation through spherical micro-indentation. Rats were exposed to a single dose of 0, 5, and 25 cGy whole body helium-4 radiation. Animals were euthanized at 7, 30, 90, or 180-days after exposure. Spherical micro-indentation was performed on axial cross sections of the femur cortical bone to determine instantaneous and relaxed shear moduli. At 90-days after exposure, the 25 cGy exposure caused a significant decline in shear modulus compared to control and 5 cGy groups. The instantaneous modulus decreased 33% and the relaxed modulus decreased 32% as compared to the sham group. This decline was followed by a recovery of both moduli, which was observed by 180-days after exposure; at 180 days, the moduli were no longer statistically different from those at 7 or 30 days. The observed decrease at 90 days, followed by recovery to baseline levels, can be attributed to the biological mechanisms involved in bone formation that were affected by radiation, bone turnover, and systemic changes in hormones due to radiation exposure. Continued assessment of the mechanisms that drive such a response in material properties may enable identification of pathways for therapeutic countermeasures against radiation exposure.

FMRP Interacts with RARα in Synaptic Retinoic Acid Signaling and Homeostatic Synaptic Plasticity.

The fragile X syndrome (FXS) is an X-chromosome-linked neurodevelopmental disorder with severe intellectual disability caused by inactivation of the fragile X mental retardation 1 (FMR1) gene and subsequent loss of the fragile X mental retardation protein (FMRP). Among the various types of abnormal synaptic function and synaptic plasticity phenotypes reported in FXS animal models, defective synaptic retinoic acid (RA) signaling and subsequent defective homeostatic plasticity have emerged as a major synaptic dysfunction. However, the mechanism underlying the defective synaptic RA signaling in the absence of FMRP is unknown. Here, we show that RARα, the RA receptor critically involved in synaptic RA signaling, directly interacts with FMRP. This interaction is enhanced in the presence of RA. Blocking the interaction between FMRP and RARα with a small peptide corresponding to the critical binding site in RARα abolishes RA-induced increases in excitatory synaptic transmission, recapitulating the phenotype seen in the Fmr1 knockout mouse. Taken together, these data suggest that not only are functional FMRP and RARα necessary for RA-dependent homeostatic synaptic plasticity, but that the interaction between these two proteins is essential for proper transcription-independent RA signaling. Our results may provide further mechanistic understanding into FXS synaptic pathophysiology.

A Mouse Model for Skeletal Structure and Function Changes Caused by Radiation Therapy and Estrogen Deficiency.

Radiation therapy and estrogen deficiency can damage healthy bone and lead to an increased fracture risk. The goal of this study is to develop a mouse model for radiation therapy using a fractionated biologically equivalent dose for cervical cancer treatment in both pre- and postmenopausal women. Thirty-two female C57BL/6 mice 13 weeks of age were divided into four groups: Sham + non-irradiated (SHAM + NR), Sham + irradiated (SHAM + IRR), ovariectomy + non-irradiated (OVX + NR) and ovariectomy + irradiated (OVX + IRR). The irradiated mice received a 6 Gy dose of X-rays to the hindlimbs at Day 2, Day 4 and Day 7 (18 Gy total). Tissues were collected at Day 35. DEXA, microCT analysis and FEA were used to quantify structural and functional changes at the proximal tibia, midshaft femur, proximal femur and L1 vertebra. There was a significant (p < 0.05) decline in proximal tibia trabecular BV/TV from (1) IRR compared to NR mice within Sham (- 46%) and OVX (- 41%); (2) OVX versus Sham within NR mice (- 36%) and IRR mice (- 30%). With homogenous material properties applied to the proximal tibia mesh using FEA, there was (1) an increase in whole bone (trabecular + cortical) structural stiffness from IRR compared to NR mice within Sham (+ 10%) and OVX (+ 15%); (2) a decrease in stiffness from OVX versus Sham within NR mice (- 18%) and IRR mice (- 14%). Fractionated irradiation and ovariectomy both had a negative effect on skeletal microarchitecture. Ovariectomy had a systemic effect, while skeletal radiation damage was largely specific to trabecular bone within the X-ray field.

AMPA receptor/TARP stoichiometry visualized by single-molecule subunit counting.

Members of the transmembrane AMPA receptor-regulatory protein (TARP) family modulate AMPA receptor (AMPA-R) trafficking and function. AMPA-Rs consist of four pore-forming subunits. Previous studies show that TARPs are an integral part of the AMPA-R complex, acting as accessory subunits for mature receptors in vivo. The TARP/AMPA-R stoichiometry was previously measured indirectly and found to be variable and dependent on TARP expression level, with at most four TARPs associated with each AMPA-R complex. Here, we use a single-molecule technique in live cells that selectively images proteins located in the plasma membrane to directly count the number of TARPs associated with each AMPA-R complex. Although individual GFP-tagged TARP subunits are observed as freely diffusing fluorescent spots on the surface of Xenopus laevis oocytes when expressed alone, coexpression with AMPA-R-mCherry immobilizes the stargazin-GFP spots at sites of AMPA-R-mCherry, consistent with complex formation. We determined the number of TARP molecules associated with each AMPA-R by counting bleaching steps for three different TARP family members: γ-2, γ-3, and γ-4. We confirm that the TARP/AMPA-R stoichiometry depends on TARP expression level and discover that the maximum number of TARPs per AMPA-R complex falls into two categories: up to four γ-2 or γ-3 subunits, but rarely above two for γ-4 subunit. This unexpected AMPA-R/TARP stoichiometry difference has important implications for the assembly and function of TARP/AMPA-R complexes.

Distinct 3'UTRs differentially regulate activity-dependent translation of brain-derived neurotrophic factor (BDNF).

Expression of the brain-derived neurotrophic factor (BDNF) is under tight regulation to accommodate its intricate roles in controlling brain function. Transcription of BDNF initiates from multiple promoters in response to distinct stimulation cues. However, regardless which promoter is used, all BDNF transcripts are processed at two alternative polyadenylation sites, generating two pools of mRNAs that carry either a long or a short 3'UTR, both encoding the same BDNF protein. Whether and how the two distinct 3'UTRs may differentially regulate BDNF translation in response to neuronal activity changes is an intriguing and challenging question. We report here that the long BDNF 3'UTR is a bona fide cis-acting translation suppressor at rest whereas the short 3'UTR mediates active translation to maintain basal levels of BDNF protein production. Upon neuronal activation, the long BDNF 3'UTR, but not the short 3'UTR, imparts rapid and robust activation of translation from a reporter. Importantly, the endogenous long 3'UTR BDNF mRNA specifically undergoes markedly enhanced polyribosome association in the hippocampus in response to pilocarpine induced-seizure before transcriptional up-regulation of BDNF. Furthermore, BDNF protein level is quickly increased in the hippocampus upon seizure-induced neuronal activation, accompanied by a robust activation of the tropomyosin-related receptor tyrosine kinase B. These observations reveal a mechanism for activity-dependent control of BDNF translation and tropomyosin-related receptor tyrosine kinase B signaling in brain neurons.

Retinoic acid-gated BDNF synthesis in neuronal dendrites drives presynaptic homeostatic plasticity.

Homeostatic synaptic plasticity is a non-Hebbian synaptic mechanism that adjusts synaptic strength to maintain network stability while achieving optimal information processing. Among the molecular mediators shown to regulate this form of plasticity, synaptic signaling through retinoic acid (RA) and its receptor, RARα, has been shown to be critically involved in the homeostatic adjustment of synaptic transmission in both hippocampus and sensory cortices. In this study, we explore the molecular mechanism through which postsynaptic RA and RARα regulates presynaptic neurotransmitter release during prolonged synaptic inactivity at mouse glutamatertic synapses. We show that RARα binds to a subset of dendritically sorted brain-derived neurotrophic factor (Bdnf) mRNA splice isoforms and represses their translation. The RA-mediated translational de-repression of postsynaptic BDNF results in the retrograde activation of presynaptic tropomyosin receptor kinase B (TrkB) receptors, facilitating presynaptic homeostatic compensation through enhanced presynaptic release. Together, our study illustrates an RA-mediated retrograde synaptic signaling pathway through which postsynaptic protein synthesis during synaptic inactivity drives compensatory changes at the presynaptic site.

Combined Hind Limb Suspension and sRANK-L on Bone Strength in Mice by Finite Element Analysis: Effects of Anatomic Model Height.

With commercial space travel on the horizon, it is important to understand how the microgravity environment of space effects bone strength. The reduction in skeletal loading is known to cause a rapid loss in bone density. How this corresponds to losses of bone strength is not well known, especially when combined with the osteoporotic effects of aging. In this study, a mouse model of hind limb suspension (HLS) was used to simulate the effects of gravitational unloading. This was combined with soluble receptor activator of nuclear factor kappa beta ligand (sRANK-L), which simulates age related osteoporosis. The proximal region of the tibia in mouse legs was scanned in-vivo pre-treatment as well as at the conclusion of the study with high resolution micro computed tomography (µCT). Subject specific finite element (FE) models were constructed from these 3D images to assess bone strength by simulating mechanical loading on these bone microstructures. Parameters indicative of bone strength obtained from the FE models were bone volume, stiffness, structural efficiency, and the 10th and 90th percentile nodal Von-Mises Stresses. Additionally, a model sensitivity analysis was performed to assess how these parameters varied with changes in anatomic model height. In regards to FE stiffness, HLS resulted in a 31% decline, sRANK-L resulted in a 16.8% decline, and HLS combined with sRANK-L (HLS+sRANK-L) resulted in a 38.6% decline. One interesting finding is that HLS caused a reduction in both bone stiffness and bone structural efficiency, while sRANK-L did not cause changes in bone structural efficiency, suggesting the importance of skeletal loading for maintaining bone health. In addition, sRANK-L combined with HLS caused an additional decline in bone stiffness, but did not further alter bone structural efficiency. In conclusion, this study shows that depending on the cause of osteoporosis, bone strength changes are not necessarily proportional to bone density changes. Thus, it is important to develop new clinical bone assessments beyond the current bone density measurement.Clinical Relevance- These parameters are associated with the microstructural mechanics of bone, and understanding how strength is decreased on a structural level may lead to the development of in-vivo bone strength testing clinically.

Chronic kidney disease and aging differentially diminish bone material and microarchitecture in C57Bl/6 mice.

Chronic kidney disease (CKD) is a common disease of aging and increases fracture risk over advanced age alone. Aging and CKD differently impair bone turnover and mineralization. We thus hypothesize that the loss of bone quality would be greatest with the combination of advanced age and CKD. We evaluated bone from young adult (6 mo.), middle-age (18 mo.), and old (24 mo.) male C57Bl/6 mice three months following either 5/6th nephrectomy, to induce CKD, or Sham procedures. CKD exacerbated losses of cortical and trabecular microarchitecture associated with aging. Aging and CKD each resulted in thinner, more porous cortices and fewer and thinner trabeculae. Bone material quality was also reduced with CKD, and these changes to bone material were distinct from those due to age. Aging reduced whole-bone flexural strength and modulus, micrometer-scale nanoindentation modulus, and nanometer-scale tissue and collagen strain (small-angle x-ray scattering [SAXS]. By contrast, CKD reduced work to fracture and variation in bone tissue modulus and composition (Raman spectroscopy), and increased percent collagen strain. The increased collagen strain burden was associated with loss of toughness in CKD. In addition, osteocyte lacunae became smaller, sparser, and more disordered with age for Sham mice, yet these age-related changes were not clearly observed in CKD. However, for CKD, larger lacunae positively correlated with increased serum phosphate levels, suggesting that osteocytes play a role in systemic mineral homeostasis. This work demonstrates that CKD reduces bone quality, including microarchitecture and bone material properties, and that loss of bone quality with age is compounded by CKD. These findings may help reconcile why bone mass does not consistently predict fracture in the CKD population, as well as why older individuals with CKD are at high risk of fragility.

The Effects of Hind Limb Suspension and Cast Mediated Immobilization on Bone Strength Properties.

Astronauts and patients on bedrest are subject to a combination of bone strength losses and muscle atrophy due to microgravity and unloading. In this study, mice were subject to both hind limb suspension and cast mediated immobilization. Pre-treatment and post-treatment microCT scans were utilized to create finite element models. Both pre-treatment and posttreatment scans were then cropped, rotated and threedimensional image registration was performed to eliminate inconsistency in alignment. A hexahedral finite element mesh was then generated from this 3D data. Finite element analysis was conducted to perform simulated physiological loading of the femoral neck to assess bone strength through bone structural morphology. Hind limb suspension combined with Cast Mediated Immobilization caused a 7.9% decrease in bone FEA stiffness compared to the in-vivo pre-treatment control. No differences were found in bone volume or structural efficiency.

Modeling Space Radiation Induced Bone Changes in Rat Femurs through Finite Element Analysis.

As the duration of manned missions outside of the Earth's protective shielding increase, astronauts are at risk for exposure to space radiation. Various organ systems may be damaged due to exposure. This study investigates the bone strength changes using finite element modeling of Long Evans rats (n=85) subjected to graded, head-only proton (0, 10, 25, and 100 cGy, 150 MeV/n) and 28silicon (0, 10, 25, and 50 cGy, 300 MeV/n) radiation. The strength of the femoral neck will be examined due its clinical relevance to hip fractures. It has been shown in previous studies that bone mineral density was not reduced at the site of fracture. These findings question whether measurements of bone mineral density may be used to assess risk of hip fracture. The mechanisms leading to the irregular relationship between bone density and strength are still uncertain within literature and investigated to greater extent in clinical applications. Finite element analysis within this study simulated physiological loading of the femoral neck. No significant changes in femoral neck strength were found across doses of proton or 28silicon head-only radiation. Future work includes performing mechanical testing of the bone samples. Moving from mouse to larger animal models may also provide the increased lifespan for assessing the long-term outcomes of radiation exposure.

Reduced Osteoarthritis Severity in Aged Mice With Deletion of Macrophage Migration Inhibitory Factor.

OBJECTIVE: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is elevated in the serum and synovial fluid of patients with osteoarthritis (OA). This study was undertaken to investigate the potential role of MIF in OA in human joint tissues and in vivo in mice with age-related and surgically induced OA. METHODS: MIF in conditioned media from human chondrocytes and meniscal cells and from cartilage explants was measured by enzyme-linked immunosorbent assay. The severity of OA was analyzed histologically in male wild-type and MIF-/- mice at 12 and 22 months of age and following destabilization of the medial meniscus (DMM) surgery in 12-week-old MIF-/- mice as well as in wild-type mice treated with a neutralizing MIF antibody. Synovial hyperplasia was graded in S100A8-immunostained histologic sections. Bone morphometric parameters were measured by micro-computed tomography. RESULTS: Human OA chondrocytes secreted 3-fold higher levels of MIF than normal chondrocytes, while normal and OA meniscal cells produced equivalent amounts. Compared to age- and strain-matched controls, the cartilage, bone, and synovium in older adult mice with MIF deletion were protected against changes of naturally occurring age-related OA. No protection against DMM-induced OA was seen in young adult MIF-/- mice or in wild-type mice treated with anti-MIF. Increased bone density in 8-week-old mice with MIF deletion was not maintained at 12 months. CONCLUSION: These results demonstrate a differential mechanism in the pathogenesis of naturally occurring age-related OA compared to injury-induced OA. The inhibition of MIF may represent a novel therapeutic target in the reduction of the severity of age-related OA.

Micromechanical modeling of calcifying human costal cartilage using the generalized method of cells.

Various tissues in the human body, including cartilage, are known to calcify with aging. There currently is no material model that accounts for the calcification in the costal cartilage, which could affect the overall structural response of the rib cage, and thus change the mechanisms and resistance to injury. The goal of this study is to investigate, through the development of a calcifying cartilage model, whether the calcification morphologies present in the costal cartilage change its effective material properties. A calcified cartilage material model was developed using the morphologies of calcifications obtained from microCT and the relaxed elastic modulus of the human costal cartilage obtained from indentation testing. The homogenized model of calcifying cartilage found that calcifications alter the effective material behavior of the cartilage, and this effect is highly dependent on the microstructural connectivity of the calcification. Calcifications which are not contiguous with the rib bone and constitute 0-18% of the cartilage volume increase the effective elastic modulus from its baseline value of 5MPa to up to 8MPa. Calcifications which are attached to the rib bone, which typically constitute 18-25% of the cartilage volume, result in effective moduli of 20-66MPa, depending on the microstructure, and introduce marked anisotropy into the material. The calcifying cartilage model developed in this study can be incorporated into biomechanical models of the aging thorax to better understand how calcifications in the aging thorax affect the structural response of the rib cage.

Synaptic retinoic acid signaling and homeostatic synaptic plasticity.

One of the defining features of the nervous system is its ability to modify synaptic strength in an experience-dependent manner. Chronic elevation or reduction of network activity activates compensatory mechanisms that modulate synaptic strength in the opposite direction (i.e. reduced network activity leads to increased synaptic strength), a process called homeostatic synaptic plasticity. Among the many mechanisms that mediate homeostatic synaptic plasticity, retinoic acid (RA) has emerged as a novel signaling molecule that is critically involved in homeostatic synaptic plasticity induced by blockade of synaptic activity. In neurons, silencing of synaptic transmission triggers RA synthesis. RA then acts at synapses by a non-genomic mechanism that is independent of its well-known function as a transcriptional regulator, but operates through direct activation of protein translation in neuronal dendrites. Protein synthesis is activated by RA-binding to its receptor RARα, which functions locally in dendrites in a non-canonical manner as an RNA-binding protein that mediate RA's effect on translation. The present review will discuss recent progress in our understanding of the novel role of RA, which led to the identification of RA as a critical synaptic signaling molecule that mediates activity-dependent regulation of protein synthesis in neuronal dendrites. This article is part of the Special Issue entitled 'Homeostatic Synaptic Plasticity'.

Morphology, distribution, mineral density and volume fraction of human calcified costal cartilage.

This study examines the properties of calcifying human costal cartilage and adjacent rib bone using qualitative and quantitative micro-computed tomography analysis. Calcifications are categorized with respect to location, microstructure, shape, and contiguity using a novel classification scheme and quantified in terms of mineral density, volume fraction, and length of infiltration from the costo-chondral junction (CCJ). Calcifications were present throughout the cartilage by location and ranged from small diffuse calcifications to nodes, rods, plates, and even large complex structures that exhibited a microstructural morphology similar to a cross-section of diaphysial bone, with a dense shell surrounding a trabecular core. Solid microstructure was most common for calcifications (44.5%), and the morphologies were found to vary with location, with rods and plates being most prevalent at the periphery (91.7% of all rods, 98.4% of all plates). The average mineral density of the calcifications over all locations and morphologies was 658.8±86.36, compared with 662.7±50.37 mgHA cm(-3) for the adjacent rib bone. The calcification volume fraction (6.54±4.71%) was less than the volume fraction of rib bone (21.62±6.44%). The length of contiguous calcification infiltrating from the CCJ into the costal cartilage, when present, was 19.21±11.65 mm. These changes in the costal cartilage should be considered in biomechanical models of the thorax since the presence, location, and morphology of the calcifications alter the material behavior of the costal cartilage, as well as the structural behavior of the entire rib.

Biomechanical response of ribs under quasistatic frontal loading.

OBJECTIVE: The goal of the present study was to identify rib-level differences in fracture characteristics for individual ribs subjected to anterior-posterior loading. METHODS: Twenty-seven individual ribs were extracted from levels 2 to 10 from 3 postmortem human subjects (2 females and one male) and subjected to anterior-posterior loading at a quasistatic (2 mm/s) loading rate. The ribs were placed in a fixture that provided a pinned boundary condition at each extremity, and each specimen was loaded to failure. Reaction force and strains on the internal and external cortical surfaces of the ribs were measured. RESULTS: Rib 2 was found to be 3 to 4 times stiffer than rib 3, whereas all other ribs were comparable in stiffness to rib 3. Fracture forces, fracture displacement, and work to fracture showed no clear rib-level trends, although the young male subject consistently exhibited higher fracture force and work values than the elderly female subjects for a given rib level. The cortical strains on the external surface of the rib remained in tension during the loading, whereas the internal surface strains were in compression. The data from the present study were compared to a similar study performed at dynamic loading rates (1.43-1.85 m/s). The quasistatic tests exhibited lower peak force and greater normalized fracture displacement than the dynamic tests, though the work was comparable between the 2 studies. CONCLUSIONS: The present study is one of the few that focuses on testing the rib as an entire structure and can contribute to understanding of how the structural behavior of an individual rib contributes to the fracture tolerance of the overall thorax when undergoing frontal loading.

Structural response of cadaveric ribcages under a localized loading: stiffness and kinematic trends.

To improve understanding of structural coupling and deformation patterns throughout the loaded ribcage, the present study reports the force-displacement and kinematic responses under a highly-localized loading condition using three PMHS ribcages (ages 44, 61, and 63 years). The ribcages were quasi-statically loaded locally to a non-failure displacement (nominally 15% of the ribcage depth at the loaded rib level) at approximately 25 unilateral locations and 5-7 geometrically symmetric bilateral locations on the anterior surface of each ribcage, for a total of 94 tests. The translations of 56 points distributed around the anterior, lateral, and posterior portions of the superficial surface of the ribcage were measured while under loading. Each of the first through sixth rib levels was then separated from the remaining ribs, and this "rib ring" structure was individually loaded at the sternum in the anterior-posterior direction. The force when the ribcage was deflected to 8% of its initial depth was normalized to the force at the upper sternum (viz. 81.2±32.2 N). The normalized unilateral force at the costo-chondral junction was found to vary from 0.76±0.29 at rib 1 to 0.15±0.02 at rib 9, while bilateral forces (sum of left and right aspects) varied from 0.92 to 1.11. The rib rings were generally less stiff, ranging from 0.78±0.24 for rib 1 to 0.19±0.01 for rib 6. The deformation patterns under all loading conditions were quantified. In general, bilateral loading produced an approximately symmetric deformation pattern, while unilateral loading resulted in approximately twice as much resultant deformation on the ipsilateral side compared to the contralateral side.

Glycosylation of beta(1)-adrenergic receptors regulates receptor surface expression and dimerization.

The beta(1)-adrenergic receptor (beta(1)AR) has one predicted site of N-linked glycosylation on its extracellular amino-terminus, but the glycosylation and potential functional importance of this site have not yet been examined. We show here that the beta(1)AR is glycosylated in various cell types and that mutation of the single predicted site of N-linked glycosylation (N15A) results in the formation of receptors that are not N-glycosylated. The beta(1)AR N15A mutant exhibited significantly decreased basal surface expression relative to the wild-type receptor but had no detectable deficits in ligand binding or agonist-promoted internalization. Co-immunoprecipitation experiments using Flag-tagged and HA-tagged receptors demonstrated that the beta(1)AR-N15A mutant receptor exhibits a markedly reduced capacity for dimerization relative to wild-type beta(1)AR. These data reveal that the beta(1)AR is glycosylated on Asn15 and that this glycosylation plays a role in regulating beta(1)AR surface expression and dimerization.

Heterodimerization of alpha 2A- and beta 1-adrenergic receptors.

beta- and alpha(2)-adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the beta(1)-adrenergic receptor (beta(1)AR) and alpha(2A)-adrenergic receptor (alpha(2A)AR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta(1)AR and alpha(2A)AR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha(2A)AR was found to induce substantial internalization of coexpressed beta(1)AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [(3)H]dihydroalprenolol binding to the beta(1)AR by various ligands revealed that beta(1)AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha(2A)AR. Finally, beta(1)AR/alpha(2A)AR heterodimerization was found to be markedly enhanced by a beta(1)AR point mutation (N15A) that blocks N-linked glycosylation of the beta(1)AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha(2A)AR. These data reveal an interaction between beta(1)AR and alpha(2A)AR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.