Growth Hormone Effects on Bone Loss-Induced by Mild Traumatic Brain Injury and/or Hind Limb Unloading.

Growth hormone (GH) deficiency and loss of physical activity are common features in traumatic brain injury (TBI) patients that may contribute to bone loss. Therefore, we tested the hypothesis that GH treatment will rescue the hind limb unloading (UL)-induced skeletal deficit in TBI mice. Mild TBI was induced once per day for four consecutive days. UL (right hind limb) and treatment (3 mg/day GH or vehicle) began two weeks after the first TBI episode and lasted for four weeks. GH treatment increased femur BMD and lean body mass but decreased the % fat measured by DXA in the Control group. Micro-CT analysis revealed that the TBI, UL and TBI-UL groups showed reduced tibia trabecular (Tb) bone mass by 15%, 70%, and 75%, respectively compared to Control mice and that GH treatment significantly increased Tb. bone mass in all four groups. Vertebra also showed reduced Tb. bone mass in TBI, UL and TBI-UL groups. GH treatment increased vertebral Tb. bone mass in Control and UL groups but not in the TBI or TBI-UL group. GH treatment increased serum IGF-I levels similarly in TBI, UL and TBI-UL groups at day 14, suggesting the GH effect on liver IGF-I production was unaffected by skeletal UL. In contrast, GH effect on expression of ALP, IGFBP5 and axin2 in bone were compromised by UL. In conclusion, skeletal UL caused a greater Tb. bone deficit than mild TBI alone and that GH anabolic effects in the TBI and UL groups vary depending on the skeletal site.

Policy options to achieve culturally-aware and environmentally-sustainable tourism in Fiji.

Increased visitation rates are expected to further impact ecosystems and local communities depending on them to generate income from tourism. We measure how different sustainable tourism management options of such areas in ways that respect the concept of vanua, the Fijian understanding of the connectiveness of the natural environment, humans and traditions, are perceived by a representative sample of potential visitors of the UK population. We then consider some plausible management options and how these may impact welfare. Results show that prospective UK respondents are willing to donate approximately £73 for a management option that enforces medium restrictions by local communities to enter coastal and marine areas in Fiji, so that vanua is respected. A management option that instead denies access to local communities is not seen favourably by prospective UK visitors to Fiji. In terms of time preference, UK respondents, in particular those with previous experiences of tropical areas, prefer environmental projects that restore and protect coastal and marine ecosystems to be completed as soon as possible. Our findings seem to support the introduction of more sustainable and community-based management practices in Fiji as they appear to increase welfare of visitors respecting local traditions and customs, as long as some access is provided to tourists. Donations from tourists or a change in tourism management from a traditional to a more sustainable practice may support the sustainable development of the local coastal communities in Fiji.

Cortical and trabecular bone are equally affected in rats with renal failure and secondary hyperparathyroidism.

BACKGROUND: Changes in mineral metabolism and bone structure develop early in the course of chronic kidney disease and at end-stage are associated with increased risk of fragility fractures. The disruption of phosphorus homeostasis leads to secondary hyperparathyroidism, a common complication of chronic kidney disease. However, the molecular pathways by which high phosphorus influences bone metabolism in the early stages of the disease are not completely understood. We investigated the effects of a high phosphorus diet on bone and mineral metabolism using a 5/6 nephrectomy model of chronic kidney disease. METHODS: Four-week old rats were randomly assigned into groups: 1) Control with standard diet, 2) Nephrectomy with standard rodent diet, and 3) Nephrectomy with high phosphorus diet. Rats underwent in vivo imaging at baseline, day 14, and day 28, followed by ex vivo imaging. RESULTS: Cortical bone density at the femoral mid-diaphysis was reduced in nephrectomy-control and nephrectomy-high phosphorus compared to control rats. In contrast, trabecular bone mass was reduced at both the lumbar vertebrae and the femoral secondary spongiosa in nephrectomy-high phosphorus but not in nephrectomy-control. Reduced trabecular bone volume adjusted for tissue volume was caused by changes in trabecular number and separation at day 35. Histomorphometry revealed increased bone resorption in tibial secondary spongiosa in nephrectomy-control. High phosphorus diet-induced changes in bone microstructure were accompanied by increased serum parathyroid hormone and fibroblast growth factor 23 levels. CONCLUSION: Our study demonstrates that changes in mineral metabolism and hormonal dysfunction contribute to trabecular and cortical bone changes in this model of early chronic kidney disease.

Experimental repetitive mild traumatic brain injury induces deficits in trabecular bone microarchitecture and strength in mice.

To evaluate the long-term consequence of repetitive mild traumatic brain injury (mTBI) on bone, mTBI was induced in 10-week-old female C57BL/6J mice using a weight drop model, once per day for 4 consecutive days at different drop heights (0.5, 1 and 1.5 m) and the skeletal phenotype was evaluated at different time points after the impact. In vivo micro-CT (µ-CT) analysis of the tibial metaphysis at 2, 8 and 12 weeks after the impact revealed a 5%-32% reduction in trabecular bone mass. Histomorphometric analyses showed a reduced bone formation rate in the secondary spongiosa of 1.5 m impacted mice at 12 weeks post impact. Apparent modulus (bone strength), was reduced by 30% (P<0.05) at the proximal tibial metaphysis in the 1.5 m drop height group at 2 and 8 weeks post impact. Ex vivo µ-CT analysis of the fifth lumbar vertebra revealed a significant reduction in trabecular bone mass at 12 weeks of age in all three drop height groups. Serum levels of osteocalcin were decreased by 22%, 15%, and 19% in the 0.5, 1.0 and 1.5 m drop height groups, respectively, at 2 weeks post impact. Serum IGF-I levels were reduced by 18%-32% in mTBI mice compared to contro1 mice at 2 weeks post impact. Serum osteocalcin and IGF-I levels correlated with trabecular BV/TV (r2 =0.14 and 0.16, P<0.05). In conclusion, repetitive mTBI exerts significant negative effects on the trabecular bone microarchitecture and bone mechanical properties by influencing osteoblast function via reduced endocrine IGF-I actions.

Thyroid hormone receptor-ß1 signaling is critically involved in regulating secondary ossification via promoting transcription of the Ihh gene in the epiphysis.

Thyroid hormone (TH) action is mediated through two nuclear TH receptors, THRα and THRß. Although the role of THRα is well established in bone, less is known about the relevance of THRß-mediated signaling in bone development. On ther basis of our recent finding that TH signaling is essential for initiation and formation of secondary ossification center, we evaluated the role of THRs in mediating TH effects on epiphysial bone formation. Two-day treatment of TH-deficient Tshr(-/-) mice with TH increased THRß1 mRNA level 3.4-fold at day 7 but had no effect on THRα1 mRNA level at the proximal tibia epiphysis. Treatment of serum-free cultures of tibias from 3-day-old mice with T3 increased THRß1 expression 2.1- and 13-fold, respectively, at 24 and 72 h. Ten-day treatment of Tshr(-/-) newborns (days 5-14) with THRß1 agonist GC1 at 0.2 or 2.0 µg/day increased BV/TV at day 21 by 225 and 263%, respectively, compared with vehicle treatment. Two-day treatment with GC1 (0.2 µg/day) increased expression levels of Indian hedgehog (Ihh) 100-fold, osterix 15-fold, and osteocalcin 59-fold compared with vehicle at day 7 in the proximal tibia epiphysis. Gel mobility shift assay demonstrated that a putative TH response element in the distal promoter of mouse Ihh gene interacted with THRß1. GC1 treatment (1 nM) increased Ihh distal promoter activity 20-fold after 48 h in chondroctyes. Our data suggest a novel role for THRß1 in secondary ossification at the epiphysis that involves transcriptional upregulation of Ihh gene.

The negative impact of traumatic brain injury (TBI) on bone in a mouse model.

INTRODUCTION: While it is well established that the brain produces hypothalamic hormones and neuropeptides that influence skeletal metabolism, the impact of traumatic brain injury (TBI) on bone is unknown. Based on the recognition from clinical studies that there is an association between TBI and long-term hypothalamic pituitary dysfunction, it was hypothesized that TBI exerts a negative impact on skeletal growth and maintenance. METHODS: To test the hypothesis, this study employed a repetitive weight drop model for TBI. Four impacts were applied for four consecutive days on 5-week old female C57BL/6 J mice. Bone measurements were taken 2 weeks after the first impact. RESULTS: Bone mineral content (BMC), bone area (B area) and bone mineral density (BMD) in the total body were reduced by 14.5%, 9.8% and 5.2%, respectively, in the impacted vs. control mice. There was a 17.1% reduction in total volumetric BMD (vBMD) and a 4.0% reduction in material vBMD in cortical bone. In trabecular bone, there was a 44.0% reduction in BV/TV. Although there was no change in the cross-sectional bone size, the tibial growth plate and the tibia itself were shortened. CONCLUSION: The repetitive animal TBI model produced an immediate, strong negative impact on bone mass acquisition in young mice.

The negative impact of single prolonged stress (SPS) on bone development in mice.

Posttraumatic stress disorder (PTSD) disrupts hypothalamic-pituitary-adrenal (HPA) axis function. Given the established role of HPA axis hormones in regulating bone metabolism, we tested the hypothesis that traumatic stress has a negative impact on bone development. We employed a variant single prolonged stress (SPS) model in which several stressors were applied to three week old C57BL/6J mice. Compared to the controls, the stressed mice showed increased freezing behavior reminiscent of PTSD symptoms. At two weeks, bone mineral content (BMC), bone area (B area) and bone mineral density (BMD) in total body based on dual-energy X-ray absorptiometry (DXA) analysis were reduced by 10.2%, 7.0% and 3.6%, respectively. Micro-CT analysis of the metaphyseal region of the excised tibia revealed that SPS caused a deterioration of trabecular architecture with trabecular number (Tb.N), BV/TV, connectivity density (Conn-Den) decreasing 12.0%, 18.9%, 23.3% and trabecular spacing (Tb.Sp), structure model index (SMI) increasing 13.9%, 21.8%, respectively. Mechanical loading increased the cross-sectional area in the mid-shaft region of the loaded right versus unloaded left tibia by 7.6% in the controls, and 10.0% in the stressed mice. Therefore, SPS applied to pre-pubertal young mice produced strong negative impact on both bone mass acquisition and trabecular architecture. Mechanical loading can be employed to increase bone size, a parameter related to bone strength, in normal as well as stressed conditions.

Lasting consequences of traumatic events on behavioral and skeletal parameters in a mouse model for post-traumatic stress disorder (PTSD).

BACKGROUND: Post-traumatic stress disorder (PTSD) is an anxiety disorder that not only affects mental health, but may also affect bone health. However, there have been no studies to examine the direct relationship between PTSD and bone. METHODOLOGY/PRINCIPAL FINDINGS: We employed electric shocks in mice to simulate traumatic events that cause PTSD. We also injected the anxiogenic drug FG-7142 prior to electric shocks. Electric shocks created lasting conditioned fear memory in all mice. In young mice, electric shocks elicited not only behavioral response but also skeletal response, and injection of FG-7142 appeared to increase both types of response. For example in behavioral response within the first week, mice shocked alone froze an average of 6.2 sec in 10 sec tests, and mice injected with FG-7142 froze 7.6 sec, both significantly different (P<0.05) from control mice, which only froze 1.3 sec. In skeletal response at week 2, shocks alone reduced 6% bone mineral content (BMC) in total body (P = 0.06), while shocks with FG-7142 injection reduced not only 11% BMC (P<0.05) but also 6% bone mineral density (BMD) (P<0.05). In addition, FG-7142 injection also caused significant reductions of BMC in specific bones such as femur, lumbar vertebra, and tibia at week 3. Strong negative correlations (R(2) = -0.56, P<0.05) and regression (y = 0.2527-0.0037 * x, P<0.01) between freezing behavior and total body BMC in young mice indicated that increased contextual PTSD-like behavior was associated with reduced bone mass acquisition. CONCLUSIONS/SIGNIFICANCE: This is the first study to document evidence that traumatic events induce lasting consequences on both behavior and skeletal growth, and electric shocks coupled with injection of anxiogenic FG-7142 in young mice can be used as a model to study the effect of PTSD-like symptoms on bone development.

The Concept of Divergent Targeting through the Activation and Inhibition of Receptors as a Novel Chemotherapeutic Strategy: Signaling Responses to Strong DNA-Reactive Combinatorial Mimicries.

Recently, we reported the combination of multitargeted ErbB1 inhibitor-DNA damage combi-molecules with OCT in order to downregulate ErbB1 and activate SSTRs. Absence of translation to cell kill was believed to be partially due to insufficient ErbB1 blockage and DNA damage. In this study, we evaluated cell response to molecules that damage DNA more aggressively and induce stronger attenuation of ErbB1 phosphorylation. We used three cell lines expressing low levels (U87MG) or transfected to overexpress wildtype (U87/EGFR) or a variant (U87/EGFRvIII) of ErbB1. The results showed that Iressa ± HN2 and the combi-molecules, ZRBA4 and ZR2003, significantly blocked ErbB1 phosphorylation in U87MG cells. Addition of OCT significantly altered cell cycle distribution. Analysis of the DNA damage response pathway revealed strong upregulation of p53 by HN2 and the combi-molecules. Apoptosis was only induced by a 48 h exposure to HN2. All other treatments resulted in cell necrosis. This is in agreement with Akt-Bad pathway activation and survivin upregulation. Despite strong DNA damaging properties and downregulation of ErbB1 phosphorylation by these molecules, the strongest effect of SSTR activation was on cell cycle distribution. Therefore, any enhanced antiproliferative effects of combining ErbB1 inhibition with SSTR activation must be addressed in the context of cell cycle arrest.

Is IL-6 the best pro-inflammatory biomarker of clinical outcomes of cancer cachexia?

BACKGROUND & AIMS: Despite the descriptive presence of cancer cachexia (CC) in clinical practice, the underlying mechanisms and diagnostic definition have not been clearly identified. Recent work, attempting to establish diagnostic and staging criteria for CC, has identified IL-6 as a biomarker. This study aimed to investigate the clinical relevance of plasma levels of four pro-inflammatory cytokines (IL-6, IL-1ß, IL-8 and TNF-α) in advanced cancer patients (ACP) to further establish their potential in the diagnostic definition of CC. METHODS: Blood was obtained from 83 ACP (47 male and 36 female, aged 34-85 years) and analyzed for white blood cells, lymphocytes, C-reactive protein, albumin and cytokines. Subjects completed questionnaires to establish weakness, loss of appetite, fatigue, quality of life and weight loss; completed tests to determine strength, body composition and sarcopenia; and consented to chart review to calculate survival and total days admitted to hospital. RESULTS: This study shows that, in ACP, IL-1ß is better associated with clinical features of the cachectic condition, such as weakness, loss of appetite, weight loss and sarcopenia, than IL-6. CONCLUSION: IL-6 may not best represent the clinical correlates of CC in ACP. Additional cytokines should be considered in the definition of this condition.

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling.

Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.

Dissociation of epidermal growth factor receptor and ErbB2 heterodimers in the presence of somatostatin receptor 5 modulate signaling pathways.

Epidermal growth factor through the stimulation of epidermal growth factor receptor (EGFR) plays a critical role in the activation of MAPKs and phosphatidylinositol-3-protein kinase/AKT cell survival pathways attributed in many pathological conditions. At the cellular level, such functions involve EGFR overactivation and phosphorylation. In the present study, we describe that human embryonic kidney-293 cells transfected with somatostatin (SST) receptor 5 (SSTR5) exhibit inhibition of EGFR phosphorylation and modulate MAPK and phosphatidylinositol-3-protein kinase/AKT cell survival signaling. Furthermore, suppression of EGFR by using small interference RNA and an antagonist (AG1478) potentiates the SST effect via activation of SSTR5 on signaling molecules. In wild-type human embryonic kidney-293 cells, EGFR/ErbB2 exists as constitutive heterodimers. The presence of SSTR5 leads to the dissociation of the heteromeric complex of EGFR/ErbB2 and display preferential heterodimerization between SSTR5 and EGFR in an agonist-dependent manner. These findings highlight a new undiscovered mechanism and potential role of SSTR5 to attenuate the EGFR-mediated signaling pathways involved in tumorigenesis. Our data indicate that the activation and/or overexpression of SST receptors along with the inhibition of EGFR will serve as an important therapeutic approach in the treatment of ErbB-positive tumors.

Somatostatin receptors 1 and 5 heterodimerize with epidermal growth factor receptor: agonist-dependent modulation of the downstream MAPK signalling pathway in breast cancer cells.

The role of somatostatin (SST) and epidermal growth factor (EGF) in breast cancer is undisputed; however, the molecular mechanisms underlying their antiproliferative or proliferative effects are not well understood. We initially confirmed that breast tumour tissues express all five somatostatin receptors (SSTR1-5) and four epidermal growth factor receptors (ErbB1-4). Subsequently, to gain insight into the function of SSTRs and ErbBs in oestrogen receptor (ER)-positive (MCF-7) or ERalpha-negative (MDA-MB-231) breast cancer cells, we defined SSTR1, SSTR5 and ErbB1 mRNA and protein expression in these two tumour cell lines. Consistent with previous studies showing SSTR1/SSTR5 heterodimerization and having seen cell-specific and ligand-selective alterations in receptor expression, we next elucidated whether SSTR1 and SSTR5 functionally interact with ErbB1 using pbFRET analysis. We subsequently determined the effects of SST and EGF either alone, or in combination, on selected downstream signalling molecules such as erk1/2, p38 and JNK. Here, we showed that both SST and EGF influenced erk1/2 phosphorylation and that SST modulated the effects of EGF in a cell-specific manner. We also demonstrated agonist-, time and cell-dependent regulation of p38 phosphorylation. We further investigated modulation of Grb2, SOS, Shc, SH-PTP1 and SH-PTP2. ErbB1 adaptor proteins known to play a role in MAPK activation, Shc, Grb2 and SOS, changed in an agonist- and cell-specific manner whereas, SH-PTP1 and SH-PTP2, adaptor proteins reported to interact with SSTRs, translocated from the cytosol to membrane in a cell-specific manner following SST and/or EGF treatment. Although several previous studies have shown crosstalk between RTKs and GPCRs, there are no reports describing SSTR (GPCR) modulation of ErbBs (RTK) in breast cancer. To the best of our knowledge, this is the first report describing crosstalk/interactions between SSTRs and ErbBs.

Biology of somatostatin in breast cancer.

The biological effects of the neuropeptide somatostatin (SST) are mediated via a family of five somatostatin receptors (SSTRs) belonging to a family of G-protein-coupled receptors (GPCRs). SSTR regulate the secretion of hormones, growth factors, neurotransmission and cell growth in receptor-specific manner. In addition, SST plays an inhibitory role in several mammary cancer models. These effects are mediated both indirectly through inhibition of hormones and growth factors which promote tumor growth as well as directly via SSTRs present on tumor cells to inhibit mitogenic signaling of growth factor receptor kinases leading to growth arrest and induction of apoptosis. Here, we present an overview on the role of SST and its analogs in breast cancer.

Heterooligomerization of human dopamine receptor 2 and somatostatin receptor 2 Co-immunoprecipitation and fluorescence resonance energy transfer analysis.

Somatostatin and dopamine receptors are well expressed and co-localized in several brain regions, suggesting the possibility of functional interactions. In the present study we used a combination of pharmacological, biochemical and photobleaching fluorescence resonance energy transfer (pbFRET) to determine the functional interactions between human somatostatin receptor 2 (hSSTR2) and human dopamine receptor 2 (hD2R) in both co-transfected CHO-K1 or HEK-293 cells as well as in cultured neuronal cells which express both the receptors endogenously. In monotransfected CHO-K1 or HEK-293 cells, D2R exists as a preformed dimer which is insensitive to agonist or antagonist treatment. In control CHO-K1 cells stably co-transfected with hD2R and hSSTR2, relatively low FRET efficiency and weak expression in co-immunoprecipitate from HEK-293 cells suggest the absence of preformed heterooligomers. However, upon treatment with selective ligands, hD2R and hSSTR2 exhibit heterodimerization. Agonist-induced heterodimerization was accompanied by increased affinity for dopamine and augmented hD2R signalling as well as prolonged hSSTR2 internalization. In contrast, cultured striatal neurons display constitutive heterodimerization between D2R and SSTR2, which were agonist-independent. However, heterodimerization in neurons was completely abolished in the presence of the D2R antagonist eticlopride. These findings suggest that hD2R and hSSTR2 operate as functional heterodimers modulated by ligands in situ, which may prove to be a useful model in designing new therapeutic drugs.

Colocalization of somatostatin receptors and epidermal growth factor receptors in breast cancer cells.

BACKGROUND: Somatostatin receptor (SSTR) expression is positively correlated with tumor size and inversely correlated with epidermal growth factor receptor (ErbB) levels and tumor differentiation. In the present study, we compared SSTR1-5 and ErbB1-4 mRNA and protein expression in two breast cancer cell lines: MCF-7 (ER+) and MDA-MB-231 (ERalpha-). RESULTS: All five SSTRs and four ErbBs were variably expressed as both cell surface and cytoplasmic proteins. In both cell lines, SSTR4 and SSTR1 were highly expressed, followed by SSTR2 and SSTR5 with SSTR3 being the least expressed subtype, at the protein level. ErbBs were variably expressed with ErbB1 as the predominant subtype in both cell lines. ErbB1 is followed by ErbB3, ErbB2 and ErbB4 in MCF-7 at both the protein and mRNA levels. In MDA-MB-231 cells, ErbB1 is followed by ErbB2, ErbB4 and ErbB3. Our results indicate significant correlations at the level of mRNA and protein expression in a cell and receptor-specific manner. Using indirect immunofluorescence, we found that, in MCF-7 cells, SSTR5 was the most prominent subtype coexpressed with ErbBs followed by SSTR3, SSTR4, SSTR1 and SSTR2, respectively. In MDA-MB-231 cells, SSTR1 colocalized strongly with ErbBs followed by SSTR5, SSTR4, SSTR3 and SSTR2. ErbBs displayed higher levels of colocalization amongst themselves in MCF-7 cells than in MDA-MB-231 cells. CONCLUSION: These findings may explain the poor response to endocrine therapy in ER-cancer. Differential distribution of SSTR subtypes with ErbBs in breast cancer cells in a receptor-specific manner may be considered as a novel diagnosis for breast tumors.

Agonist-induced up-regulation of human somatostatin receptor type 1 is regulated by beta-arrestin-1 and requires an essential serine residue in the receptor C-tail.

We have previously shown that the human somatostatin receptor type 1 (hSSTR1) does not undergo agonist-induced internalization, but is instead up-regulated at the membrane upon prolonged somatostatin (SST) exposure. The deletion of the carboxyterminal C-tail of the receptor completely abolishes up-regulation. To identify molecular signals that mediate hSSTR1 up-regulation, we created mutant receptors with progressive C-tail deletions. Up-regulation was found to be absent in mutants lacking residues Lys359-Ser360-Arg361. Moreover, point mutation of Ser360 to Ala completely abolished up-regulation. The coexpression of wild type hSSTR1 with V53D, a dominant negative mutant of beta-arrestin-1, completely blocked hSSTR1 up-regulation. Further analysis demonstrated that calcium-calmodulin (CaM) dependent kinases were essential for the SST-induced up-regulation response. Like wild type receptors, all mutants failed to internalize after agonist exposure and were able to inhibit forskolin-stimulated cAMP accumulation. Taking these data together, we suggest that SST-induced hSSTR1 up-regulation is critically dependent upon a specific Lys-Ser-Arg sequence in the C-tail of the receptor, with Ser360 being essential. Up-regulation also requires the participation of CaM protein kinases and interactions with beta-arrestins. In contrast, coupling to adenyl cyclase (AC) and internalization occur independently of molecular signals in the receptor's C-tail.