G protein-coupled receptor-mediated autophagy in health and disease.

G protein-coupled receptors (GPCRs) constitute the largest and most diverse superfamily of mammalian transmembrane proteins. These receptors are involved in a wide range of physiological functions and are targets for more than a third of available drugs in the market. Autophagy is a cellular process involved in degrading damaged proteins and organelles and in recycling cellular components. Deficiencies in autophagy are involved in a variety of pathological conditions. Both GPCRs and autophagy are essential in preserving homeostasis and cell survival. There is emerging evidence suggesting that GPCRs are direct regulators of autophagy. Additionally, autophagic machinery is involved in the regulation of GPCR signalling. The interplay between GPCR and autophagic signalling mechanisms significantly impacts on health and disease; however, there is still an incomplete understanding of the underlying mechanisms and therapeutic implications in different tissues and disease contexts. This review aims to discuss the interactions between GPCR and autophagy signalling. Studies on muscarinic receptors, beta-adrenoceptors, taste receptors, purinergic receptors and adhesion GPCRs are summarized, in relation to autophagy.

Exploring orphan GPCRs in neurodegenerative diseases.

Neurodegenerative disorders represent a significant and growing health burden worldwide. Unfortunately, limited therapeutic options are currently available despite ongoing efforts. Over the past decades, research efforts have increasingly focused on understanding the molecular mechanisms underlying these devastating conditions. Orphan receptors, a class of receptors with no known endogenous ligands, emerge as promising druggable targets for diverse diseases. This review aims to direct attention to a subgroup of orphan GPCRs, in particular class A orphans that have roles in neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Multiple sclerosis. We highlight the diverse roles orphan receptors play in regulating critical cellular processes such as synaptic transmission, neuronal survival and neuro-inflammation. Moreover, we discuss the therapeutic potential of targeting orphan receptors for the treatment of neurodegenerative disorders, emphasizing recent advances in drug discovery and preclinical studies. Finally, we outline future directions and challenges in orphan receptor research.

Regulation of M2, M3, and M4 muscarinic receptor expression in K562 chronic myelogenous leukemic cells by carbachol.

CONTEXT: Muscarinic receptors mediate a variety of cellular responses to acetylcholine, including inhibition of adenylate cyclase, breakdown of phosphoinositide and modulation of ion channels. These receptors are relatively abundant in the central nervous system and peripheral parasympathetic nervous system. Many cells express a mixture of muscarinic receptor transcripts. Changes in muscarinic M(2) and M(3) receptor mRNA levels in response to agonist treatment have been reported in cerebellar granule cells, Chinese hamster ovary cells, lymphocytes and in the human neuroblastoma cell line SH-SY5Y. OBJECTIVE: In this study, we investigated the effects of agonist stimulation on cell proliferation and on the levels of muscarinic receptor expression in K562 chronic myelogenous leukemia cells. METHODS: Total RNA and crude membrane fractions were prepared from K562 cells challenged with carbachol (CCh). Muscarinic receptor subtype expression was determined by RT-PCR and western blot analysis. Proliferation and cell viability were evaluated by the trypan blue exclusion test and BrDU labeling. RESULTS: We showed that CCh-treatment leads to changes in muscarinic M(2), M(3), and M(4) receptor transcripts as well as M(2) and M(3) protein levels. We also found that CCh decreased proliferation of K562 cells in a time dependent manner, an effect prevented by atropine. These results suggest that CCh modulates K562 chronic myelogenous leukemic cells proliferation through muscarinic acetylcholine receptors.

The role of G protein ß3 subunit polymorphisms C825T, C1429T, and G5177A in Turkish subjects with essential hypertension.

Hypertension is a multifactorial disorder that constitutes a major risk factor for the cardiovascular system. Heterotrimeric G-proteins, which couple receptors for diverse extracellular enzymes or ion channels, are correlated with disease mechanisms. Several studies have demonstrated an association between G protein polymorphisms and essential hypertension in some populations, although contradictive results also exist. In this study, we have investigated the potential role of the C825T, C1429T, and G5177A polymorphisms of the ß3 subunit of G-proteins in essential hypertension in a group of Turkish subjects. Genomic DNA from 106 normotensive individuals (117.4 ± 13.1, 75.2 ± 10.5; systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels, respectively) and 101 hypertensive subjects (152.3 ± 18.0, 92.5 ± 11.6; SBP and DBP levels, respectively) were studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods for these polymorphisms. Allele frequencies of the polymorphisms were consistent with Hardy Weinberg equilibrium, except for the C825T polymorphism (χ(2) = 7.8). The frequencies of the 825T and 1429T variants were higher in hypertensive subjects compared to those of controls. Differences between hypertensives and controls were not statistically significant, though difference was very close to significance for C825T (p = 0.056 and 0.099 for 825T and 1429T, respectively). T allele frequency in overall population showed significant association with hypertension for C825T (0.0134). The prevalence of the 5177A-variant was very low and all subjects carrying it were heterozygotes in both groups.

Investigation of the association between dopamine D1 receptor gene polymorphisms and essential hypertension in a group of Turkish subjects.

Dopamine has been shown to influence blood pressure by regulating renal sodium excretion through direct interaction with the dopamine receptors, especially with the Dopamine D1 receptor (DRD1). To better understand the role of polymorphisms in those effects, we investigated the association between two polymorphic sites in the DRD1 promoter region (A-48G, G-94A) and essential hypertension in the Turkish population. The DRD1 variants were genotyped by restriction fragment length polymorphism (RFLP) analysis. A total of 205 unrelated individuals were enrolled in the study. We found that genotype distributions and allele frequencies of the control and hypertensive subjects were very similar and did not show any significant difference with respect to blood pressure (BP) and hypertension. Contribution of the gene variances in BP or hypertension by sex differences and dependence on body mass index (BMI) were also evaluated. Distribution of genotypes and allele frequencies were found to be in line with previous reports. However, increments detected in hypertensive subjects were far from being statistically significant.

M2, M3, and M4 muscarinic receptors are expressed in the guinea pig gallbladder.

AIM: The identity of muscarinic acetylcholine receptors (mAchR) involved in cholinergic-mediated contraction of the guinea pig gallbladder has been a matter of debate. Different groups have suggested the involvement of M(1), M(2), M(3), or M(4) receptor subtypes in the contraction of this tissue. The objective of this study was to identify the mAchR subtypes expressed in the guinea pig gallbladder by RT-PCR. METHODS: Total RNA prepared from frozen guinea pig gallbladder tissue was amplified by using specific primers for the M(1)-M(4) receptor subtypes. RESULTS: M(2), M(3), and M(4) transcripts were detected in the following rank order: M(4) > M(2) > M(3). We were unable to demonstrate the expression of the M(1) receptor subtype in this tissue. CONCLUSIONS: Our results are in agreement with our previous binding and functional data.

Role of G proteins and ERK activation in hemin-induced erythroid differentiation of K562 cells.

Heterotrimeric G proteins which couple extracellular signals to intracellular effectors play a central role in cell growth and differentiation. The pluripotent erythroleukemic cell line K562 that acquires the capability to synthesize hemoglobin in response to a variety of agents can be used as a model system for erythroid differentiation. Using Western blot analysis and RT-PCR, we studied alterations in G protein expression accompanying hemin-induced differentiation of K562 cells. We demonstrated the presence of G(alpha s), G(alpha i2) and G(alpha q) and the absence of G(alpha i1), G(alpha o) and G(alpha 16) in K562 cells. We observed the short form of G(alpha s) to be expressed predominantly in these cells. Treatment of K562 cells with hemin resulted in an increase in the levels of G(alpha s) and G(alpha q). On the other hand, the level of G(alpha i2) was found to increase on the third day after induction with hemin, followed by a decrease to levels lower of those of uninduced cells. The mitogen-activated protein kinase ERK1/2 pathway is crucial in the control of cell proliferation and differentiation. Both Gi- and Gq-coupled receptors stimulate MAPK activation. We therefore examined the phosphorylation of ERK1/2 during hemin-induced differentiation of K562 cells. Using anti-ERK1/2 antibodies, we observed that ERK2 was primarily phosphorylated in K562 cells. ERK2 phosphorylation increased gradually until 48 h and returned to basal values by 96 h following hemin treatment. Our results suggest that changes in G protein expression and ERK2 activity are involved in hemin-induced differentiation of K562 cells.

Methoctramine and gallamine inhibit PI hydrolysis in guinea-pig gallbladder.

The present study aimed to determine the effect of two M2/M4-selective muscarinic receptor antagonists on blocking the hydrolysis of carbachol (CCh) stimulated phospho-inositide (PI) breakdown in order to address the possibility that a muscarinic receptor other than the M(3) receptor is involved in PI hydrolysis in this tissue. Gallbladder tissue slices labeled with myo-[2-3H] inositol were incubated with increasing concentrations of antagonists and agonist. After the reactions were terminated by the addition of chloroform/methanol, labeled inositol phosphates were separated using anion exchange chromatography. Muscarinic M2 antagonists methoctramine and gallamine both inhibited carbachol-induced PI breakdown at high concentrations, with log IC50 values of -5.145 and -6.049, respectively. Gallamine at 10(-5)M concentration failed to displace the dose-response curve for carbachol-induced accumulation of inositol triphosphate (IP3). Our data suggest that M(3) receptors play a major role in stimulation of PI hydrolysis in the guinea-pig gallbladder.

The role of intracellular pathways in the proliferation of human K562 cells mediated by muscarinic receptors.

Muscarinic acetylcholine receptors (mAChRs) are members of the superfamily of G protein coupled receptors (GPCRs). Muscarinic receptors are relatively abundant in the central nervous system and in the peripheral parasympathetic nervous system. Several studies have suggested that muscarinic receptors also mediate some cellular events in hematopoietic cells. K562 erythroleukemia cells contain muscarinic receptors M2, M3 and M4, and activation of muscarinic receptors changes cell proliferation. We examined the effects of several compounds on cell proliferation in K562 erythroleukemia cells. These included a muscarinic receptor agonist carbachol (CCh), a protein kinase inhibitor staurosporine; the phospholipase C inhibitor U73122, the MEK 1-2 inhibitor UO126, the PI3-kinase inhibitor wortmannin, the Ca(2+) chelators BAPTA/AM and 2-aminoethoxy-diphenylborate (2APB). In addition, we also investigated muscarinic receptor mediated protein kinase C (PKC) expression in K562 cells. CCh caused a decrease in DNA synthesis in K562 cells supplemented with 1% fetal bovine serum after starvation. Pre-treatment of K562 cells with U73122 and BAPTA/AM antagonized the inhibitory effect of CCh, suggesting that phospholipase C and intracellular calcium are involved in CCh-mediated inhibition of proliferation in K562 cells. Our data also suggest that the regulatory roles of protein kinase C and the MAPK/ERK pathways in K562 cell proliferation are independent of cholinergic activation.

Prognostic role of sensitive-to-apoptosis gene expression in rectal cancer.

AIM: To investigate the association between prognosis of rectal cancer treated with chemoradiotherapy (CRT) and expression of sensitive-to-apoptosis (SAG), B-cell lymphoma-extra large (Bcl-X(L)) and Bcl-2 homologous antagonist/killer (Bak). METHODS: Real-time quantitative polymerase chain reaction was used to determine the expression of proteins of interest, namely SAG, Bcl-X(L), Bak and ß-actin, in rectal carcinoma patients who had a follow-up period of 3 years after CRT. Biopsy specimens were excised from the rectal tumor preceding CRT. RESULTS: SAG, Bcl-X(L) and Bak proteins showed significant correlations with each other. In multivariate analysis, patients with high vs low SAG expression showed a statistically significant difference in 2-year survival rates: 56% vs 73%, respectively (P = 0.056). On the other hand, there were no significant correlations between the expression levels of all three genes and metastatic rates or tumor responses to CRT. Mean overall survival in the patients with elevated SAG expression was 27.1 mo ± 3.9 mo [95% confidence interval (CI): 19.3-34.9], and in patients with reduced expression, it was 32.1 mo ± 2.5 mo (95% CI: 27.3-36.9). The corresponding values for Bcl-X(L) were 28.0 mo ± 4.1 mo (95% CI: 19.9-36.1) and 31.7 mo ± 2.9 mo (95% CI: 26.0-37.5), and those for Bak were 29.8 mo ± 3.7 mo (95% CI: 22.5-37.2) and 30.6 mo ± 2.4 mo (95% CI: 25.5-35.0), respectively. CONCLUSION: Two-year survival rates significantly correlated with low SAG expression, and SAG may be a candidate gene for good prognosis, independent of therapeutic response of different individuals.

G protein mutations in pituitary tumors: a study on Turkish patients.

Activating mutations of the G proteins, Gsalpha (gsp) and Gi2alpha (gip) have been reported in subsets of pituitary tumors. The objective of the study was to assess the frequency of gsp and gip mutations in pituitary tumors from Turkish patients and to investigate the possibility of mutations of protein kinase A catalytic subunit (PKAC) that activates the downstream effectors of adenylyl cyclase. PCR-amplified genomic DNA was analyzed for the presence of mutations in codons 201 and 227 of Gsalpha, codon 179 and 205 of Gi2alpha and codon 196 of PKAC, by single strand conformation polymorphism analysis, allele-specific oligonucleotide hybridization and DNA sequencing. Twenty-two patients from Turkey, 15 females and 7 males were investigated; 7 somatotroph adenomas, 7 clinically non-functioning tumors, 7 prolactinomas and 1 corticotroph adenoma. G protein mutations were identified in 6 of 22 (27.3%) pituitary tumors. Four tumors (3/7 somatotroph adenomas, 43%, 1/7 clinically non-functioning tumor) demonstrated gsp mutations at codon 201 arginine to cysteine and one recurrent somatotroph adenoma demonstrated a mutation of the Gi2alpha gene at codon 193 lysine to arginine. One tumor exhibited a C to T variation in the intervening sequence between codons 179 and 205 of the Gi2alpha gene. No mutations at codon 227 of Gsalpha, codons 179 and 205 of Gi2alpha and codon 196 of the PKAC gene were identified.