Contraception by induction of luteinized unruptured follicles with short-acting low molecular weight FSH receptor agonists in female animal models.

During recent decades minor innovative drugs have been developed for the female contraceptive market and they all contain steroidal progestagens (and estrogens) that act centrally and have side effects that can be attributed to this central action. In this study, we present an innovative tissue-specific approach for female contraception by low molecular weight (LMW) FSH receptor (FSHR) agonists, which interact with the FSHR that is dominantly expressed in the granulosa cells. The oral administration of LMW FSHR agonists with a short circulation time, induced formation of luteinized unruptured follicles (LUFs) from the Graafian follicles, thereby preventing the release of the oocyte. The short-acting LMW FSHR compounds were fully agonistic to FSHR (EC(50)=4-5 nM). In an isolated mouse follicle culture, a short incubation period (2 h) resulted in inhibition of follicular rupture, where continuous incubation induced follicle growth. Pharmacokinetics after oral administration showed a surge-like exposure in rats and monkeys. Oral administration of short-acting LMW FSHR agonists inhibited ovulation at 10 mg/kg in rats and guinea pigs by generating LUFs without affecting cyclicity. Also, inhibition of follicular rupture was shown to be reversible within one cycle. Finally, LUFs were induced without affecting the hormonal cyclicity in cynomolgus monkeys, a mono-ovulatory species. In healthy women LUF formation occurs naturally, with a LUF acting as corpus luteum that produces enough progesterone to ensure normal menstrual cyclicity. Together with the presented data this indicates that the innovative approach with short-acting LMW FSHR agonists could lead to oral contraception for females at the ovarian level.

Isolation, sequence and functional expression of the mouse m4 muscarinic acetylcholine receptor gene.

Molecular cloning studies have demonstrated the existence in mammals of five genes encoding distinct muscarinic acetylcholine receptors which are heterogeneously expressed in the central nervous system and the target organs of the autonomic nervous system. In order to determine the factors responsible for regulation of receptor expression, we isolated a genomic clone encoding the mouse m4 muscarinic acetylcholine receptor. The gene contains no introns in the coding sequence, and encodes a polypeptide of 479 amino acids that is able to bind various muscarinic ligands with affinities typical for mAChRs and to couple efficiently to inhibition of adenylyl cyclase.

Molecular diversity of sphingolipid signalling.

Sphingolipid breakdown products are now being recognized to play a dual role in cellular signalling, acting as intracellular as well as extracellular signalling molecules. Both types of action may even be found with one sphingolipid species. The recent demonstration of G protein-coupled receptors with high affinity for sphingosine 1-phosphate and sphingosylphosphorylcholine has been followed by the discovery of several novel sphingolipid actions, such as regulation of heart rate, oxidative burst, neurite retraction or platelet activation. Ligand profiles and concentration-response relationships suggest the existence of putative sphingolipid receptor subtypes. Against this background, several observations on supposed sphingolipid second messenger actions deserve a new evaluation.

Discrimination between plasma membrane and intracellular target sites of sphingosylphosphorylcholine.

On the background of the emerging concept of G protein-coupled sphingolipid receptors, Ca2+ mobilization by sphingosylphosphorylcholine (SPPC) in intact cells and SPPC-induced Ca2+ release in permeabilized cells, both occurring at similar, micromolar concentrations, were characterized and compared. In intact human embryonic kidney (HEK-293) cells, SPPC rapidly increased [Ca2+]i by mobilization of Ca2+ from thapsigargin-sensitive stores. In saponin-permeabilized HEK-293 cells, SPPC released stored Ca2+, in a manner similar to but independent of inositol 1,4,5-trisphosphate. Only the action of SPPC on intact cells, but not that in permeabilized cells, was, at least in part, sensitive to pertussis toxin. In addition and most important, Ca2+ release by SPPC in permeabilized cells was not stereoselective, whereas in intact cells only the naturally occurring D-erythro-SPPC, but not L-threo-SPPC, increased [Ca2+]i. Stereoselectivity of SPPC-induced [Ca2+]i increase was also demonstrated in bovine aortic endothelial cells. In conclusion, Ca2+ mobilization by SPPC in intact cells is independent of the previously described SPPC-gated Ca2+ channel on endoplasmic reticulum but probably mediated by a membrane sphingolipid receptor. Thus, SPPC can regulate Ca2+ homeostasis by acting apparently at two cellular targets, which exhibit clearly distinct recognition patterns.

Autoradiographic visualization of muscarinic receptors in pulmonary nerves and ganglia.

We investigated autoradiographically the distribution of muscarinic receptors in bovine airways using (-)-[3H]quinuclidinyl benzilate as radioligand. The autoradiographs demonstrated the presence of muscarinic receptors in smooth muscle as well as neuronal muscarinic receptors in pulmonary nerves and ganglia. It is reasonable to believe that the neuronal muscarinic receptors participate in the regulation of neurotransmitter release at the peripheral nerve terminals innervating the bronchial smooth muscle.

Agonist-induced down-regulation of the m4 muscarinic acetylcholine receptor occurs without changes in receptor mRNA steady-state levels.

The regulation of m4 muscarinic acetylcholine receptor mRNA expression by receptor activation was studied in N1E-115 neuroblastoma and AtT-20 pituitary cells that endogeneously express the m4 muscarinic receptor. Receptor concentration was measured by binding of the muscarinic receptor radioligand [3H]quinuclidinyl benzilate, and RNA-RNA solution hybridization/RNase protection assay with a m4 receptor-specific [32P]-cRNA probe was used to evaluate the levels of receptor mRNA. Treatment of both cell lines with a receptor-saturating concentration of the agonist carbachol decreased receptor number. However, there was no change in steady-state levels of m4 mAChR mRNA in both cell lines. Determination of mRNA stability in the presence of the transcription blocker actinomycin D revealed that carbachol treatment increased half-life of receptor mRNA in N1E-115 cells, but not in AtT-20 cells, suggesting that receptor activation can regulate m4 receptor mRNA stability dependently on cell type. Analysis of receptor degradation kinetics in the presence of the protein synthesis inhibitor cycloheximide showed that receptor down-regulation in N1E-115 and AtT-20 cells is sufficiently accounted for by increased receptor degradation. These results indicate than m4 muscarinic receptor down-regulation is substantially different from that of the muscarinic receptor subtypes m2 and m3 which is reported to be associated with agonist-induced reduction in receptor mRNA.

Calcium signalling by G protein-coupled sphingolipid receptors in bovine aortic endothelial cells.

Besides its role as a putative second messenger releasing Ca2+ from intracellular stores, sphingosine-1-phosphate (SPP) has recently been identified as an extracellularly acting ligand activating a high affinity G protein-coupled membrane receptor in various cell types. Since SPP can be released from activated platelets, we examined in the present study whether endothelial cells express receptors for SPP and related sphingolipids. In bovine aortic endothelial cells loaded with fura-2, addition of SPP caused a rapid and transient increase in intracellular Ca2+ concentration ([Ca2+]i), amounting to maximally about 230 nM. Removal of extracellular Ca2+ revealed that SPP-induced [Ca2+]i elevations were due to both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Pretreatment of the cells with pertussis toxin inhibited the SPP-induced increase in [Ca2+]i by 83%, in line with the previously reported involvement of G proteins of the Gi/o family in SPP signalling in other cell types. In contrast to other [Ca2+]i-elevating agonists, e.g., ATP and bradykinin, SPP did not activate phospholipase C in bovine aortic endothelial cells, suggesting the involvement of a novel, unidentified signalling pathway in SPP-induced release of intracellular Ca2+. Furthermore, SPP also did not cause activation of either phospholipase D or A2. Out of various related sphingolipids studied, only sphingosylphosphorylcholine (SPPC) induced a similar maximal increase in [Ca2+]i as SPP, and its effect was also fully pertussis toxin-sensitive. However, the potencies of the two sphingolipids to increase [Ca2+]i differed by more than two orders of magnitude, with the EC50 values being 0.8 nM and 260 nM for SPP and SPPC, respectively. These results identify SPP and SPPC as novel and potent endothelial agonists, inducing calcium signalling by activation of a Gi/o protein-coupled receptor(s). Given the recently reported release of SPP from thrombin-activated platelets, SPP may represent a novel mediator of platelet-endothelial cell interactions.

Muscarinic acetylcholine receptor trafficking in streptolysin O-permeabilized MDCK cells.

We investigated the validity of streptolysin O (SLO)-permeabilized Madin-Darbin canine kidney (MDCK) cells which express muscarinic acetylcholine receptors (mAChRs) coupled to pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) for the study of the molecular machinery that regulated mAChR internalization and recycling. Exposure of SLO-permeabilized cells to carbachol-reduced cell surface receptor number by up to 40% without changing total receptor number. The kinetics and maximal extent of receptor internalization as well as the potency of carbachol to induce receptor internalization were almost identical in SLO-permeabilized and non-permeabilized cells. Using this semi-intact cell system, we studied the effect of various agents affecting components potentially involved in receptor trafficking. Internalization was prevented by treatment of the SLO-permeabilized MDCK cells with (i) the stable ATP analogues, adenosine 5'-O-(3-thiotriphosphate) and adenylylimidodiphosphate, to block ATP-dependent processes, and (ii) heparin to block G protein-coupled receptor kinases. Inclusion of the stable GTP analogue, guanosine 5'-O-(3-thiotriphosphate), increased the rate but not the extent of receptor internalization. None of the membrane-impermeant agents affected receptor internalization in intact MDCK cells. This model system also allowed recycling of internalized receptors back to the plasma membrane. After removal of the agonist, cell surface receptor number in SLO-permeabilized cells returned to control values within 90 min with the same kinetics as seen in intact cells. Inclusion of guanosine 5'O-(3-thiotriphosphate) shortened the recovery time. These data suggest that both ATP-dependent kinases including G protein-coupled receptor kinases and G proteins participate in receptor internalization and recycling. In summary, the SLO-permeabilized MDCK cell is a feasible model system for the study of mAChR internalization and recycling and allows manipulation of the intracellular milieu with membrane-impermeable macromolecules.

Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type.

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca2+]i with EC50 values of about 2 microM and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP3) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca2+]i were 300-350 nM and mainly, almost 90%, due to influx of extracellular Ca2+. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca2+]i with EC50 values of about 20 microM and 7 microM, respectively. Maximal InsP formation was only 10-15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca2+]i were similar in both cell types. Formation of InsP3 was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca2+]i increases induced by m2 mAChR activation were exclusively due to Ca2+ mobilization from intracellular stores. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca2+]i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca2+ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.

Characterization of the muscarinic receptor in human tracheal smooth muscle.

Muscarinic receptors in human tracheal smooth muscle were characterized by radioligand binding and functional studies. Specific [3H]-(-)-quinuclidinylbenzilate ([3H]-(-)-QNB) binding to tracheal smooth muscle membranes was reversible, stereoselective and of high affinity (Kd = 47 +/- 4 pmol/l; RT = 920 +/- 120 fmol/g tissue). Inhibition of specific [3H]-(-)-QNB binding by the M-1 selective antagonist pirenzepine was found to occur at relative high concentrations classifying the muscarinic receptor population as belonging to the M-2 subclass. Inhibition of specific [3H]-(-)-QNB binding by muscarinic agonists revealed the presence of high and low affinity sites in nearly equal proportions. 5'-Guanylylimidodiphosphate converted high affinity sites into low affinity sites although its effect was minimal. Log dose-contraction curves of methacholine had Hill coefficients of 1.10 +/- 0.04 with pD2-values of 6.75 +/- 0.02. Inhibition of specific [3H]-(-)-QNB binding by methacholine, however, was best described by a two binding site model with pKi-values considerably lower. The difference between these affinity values points to the presence of substantial receptor reserve.

Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors.

Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.

Regulation of expression and function of muscarinic receptors.

The regulation of expression and function of the muscarinic acetylcholine receptor has been studied using several different systems. The role of glycosylation of the m2 receptor was examined by removal of glycosylation sites using site-directed mutagenesis followed by expression in stably transfected cells. The results demonstrated that glycosylation was not required for the synthesis and appearance of the receptors on the cell surface or for the coupling of the receptors to inhibition of adenylyl cyclase activity. Site-directed mutagenesis also was used to demonstrate that the single cysteine in the carboxy terminal domain of the m2 receptor was not required for receptor function, thus rendering unlikely a model suggesting a requirement for palmitoylation of this cysteine in receptor function. The muscarinic receptors expressed in embryonic chick heart were identified by molecular cloning. Two genes were initially identified which are expressed in chick heart and correspond to the chick m2 and m4 receptors. Experiments using the polymerase chain reaction to identify low abundance mRNAs indicate that at least one addition receptor gene is expressed in chick heart. In cell culture, activation of the muscarinic receptors decreases the levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription due to both mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. The elucidation of the factors which regulate the expression and function of muscarinic acetylcholine receptors (mAChR) is of obvious importance in understanding the mechanisms underlying cholinergic transmission. In this chapter, we will describe studies on the expression and function of wild type and mutant muscarinic receptors, the molecular characterization of mAChR expressed in chick heart, and the regulation of mAChR gene expression in response to muscarinic receptor activation.

Beta-adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation.

Specific binding of [125I]-(-)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (Kd = 5.3 +/- 0.9 pmol/l and RT = 78 +/- 7 fmol/g tissue). The beta 1-selective antagonists atenolol and LK 203-030 inhibited specific [125I]-(-)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous beta 2-adrenoceptor population. In one subject using LK 203-030 a small beta 1-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pKH- and pKL-values for the high and low affinity sites were 8.0 +/- 0.2 and 5.9 +/- 0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD2-value of 6.63 +/- 0.19.

Multiple pathways for the dynamin-regulated internalization of muscarinic acetylcholine receptors.

An important regulatory pathway of G-protein-coupled receptors (GPCRs) is the internalization of receptors into the cell interior. To unravel the molecular mechanisms by which GPCRs are internalized, we have studied the internalization of various members of the family of muscarinic acetylcholine receptors (mAChRs). Using the transient expression system of HEK-293 cells, we showed that the M(1), M(3) and M(4) mAChRs are internalized into clathrin-coated vesicles and recycle back to the plasma membrane. This internalization pathway is dependent on the concerted action of beta-arrestin, c-Src and the GTPase dynamin, which 'catalyses' the budding of clathrin-coated vesicles (and other vesicles) from the plasma membrane. Internalization of the M(2) mAChR (which is highly structurally and functionally related to the M(4) receptor subtype) also requires dynamin, but proceeds in an apparent beta-arrestin-, c-Src- and clathrin-independent manner. Internalized M(2) mAChRs also show virtually no receptor recycling, but are down-regulated. This demonstrates that GPCRs can be internalized by multiple dynamin-dependent pathways in a highly regulated manner.

Chemical modification of rat cerebral cortex M1 muscarinic receptors: role of histidyl residues in antagonist and agonist binding.

Chemical modification of muscarinic M1 receptors in a synaptoneurosomal preparation of rat cerebral cortex by a hydrophilic histidyl-group-specific reagent, diethylpyrocarbonate (DEP), reduces the number of [3H]-4NMPB binding sites in a dose-dependent way. The effect can be reversed by hydroxylamine treatment. No such effect is observed when carbethoxylation with 2.5 mM DEP is carried out in the presence of atropine, 4NMPB, pirenzepine or carbachol. These findings indicate that DEP specifically modifies histidyl residue(s) positioned at the binding site in members of the M1 receptor family. However, treatment with 2.5 mM DEP in the presence of various muscarinic ligands significantly disturbs the binding state of agonists. The results suggest that M1 receptors may have more than one histidyl residue of importance in ligand binding.

Evidence for an endogenous factor involved in maintenance of pirenzepine high-affinity binding in rat brain stem.

Sucrose gradient centrifugation was used to isolate membranes enriched in muscarinic receptors from bovine brain stem. Unlike the receptors in crude synaptosomal preparations of this tissue, the enriched preparations displayed only low-affinity pirenzepine binding. Similar results were obtained when purified preparations were preincubated for 1 hr in pH 7.0 buffer at 37 degrees C; however, preincubation in a pH 5.0 buffer partially restored the high-affinity pirenzepine binding. These results suggest that an endogenous factor, which is present in the crude synaptosomes of the brain stem and is removed by sucrose gradient centrifugation, is involved in maintenance of the high-affinity pirenzepine binding of the muscarinic receptors.

The cysteine residue in the carboxyl-terminal domain of the m2 muscarinic acetylcholine receptor is not required for receptor-mediated inhibition of adenylate cyclase.

Muscarinic acetylcholine receptors (mAChRs) share with many other receptors of the guanine nucleotide-binding protein-coupled receptor family a highly conserved cysteine residue in the putative cytoplasmic carboxyl-terminal region of the protein. Because elimination of this cysteine in the beta 2-adrenergic receptor has been reported to decrease functional responsiveness, we determined if this cysteine residue is essential for mAChR-effector coupling by replacing Cys457 of the m2 mAChR with glycine and expressing wild-type and mutant receptor in Chinese hamster ovary (CHO) cells. The mutant and wild-type receptors exhibited similar affinities for binding of muscarinic ligands. In addition, the mutation did not affect cell surface localization or receptor-mediated inhibition of adenylate cyclase. These results indicate that the cysteine residue in the carboxyl-terminal domain of the m2 mAChR is not required for ligand binding or mAChR-mediated inhibition of adenylate cyclase in CHO cells.

Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. Analysis of the role of N-glycosylation in receptor expression and function.

The cardiac m2 muscarinic acetylcholine receptor (mAChR) is a sialoglycosylated transmembrane protein which has three potential sites for N-glycosylation (namely, Asn2, Asn3, and Asn6). To investigate the role of N-linked oligosaccharide(s) in the expression and function of the receptor, we constructed glycosylation-defective mutant receptor genes in which the three asparagine codons were substituted by codons for either aspartate (Asp2,3,6), lysine (Lys2,3,6), or glutamine (Gln2,3,6). The glycosylation-defective and wild-type receptor genes were stably expressed in Chinese hamster ovary cells. Binding experiments with the membrane-permeable radioligand [3H]quinuclidinyl-benzilate and the membrane-impermeable radioligand [3H]N-methylscopolamine revealed that the Asp2,3,6, Gln2,3,6, and wild-type receptors were located exclusively on the cell surface and expressed in similar numbers. The Lys2,3,6 mutant receptor was expressed at a relatively low level and was therefore not included in subsequent experiments. Wheat germ agglutinin-Sepharose chromatography and sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis demonstrated that the wild-type receptor, but not the Asp2,3,6 and Gln2,3,6 mutant receptors were N-glycosylated. The Asp2,3,6 and Gln2,3,6 mutant receptors had the same affinities for mAChR ligands as wild-type receptors. The time courses for degradation of the Asp2,3,6, Gln2,3,6, and wild-type receptors were also similar. In vivo functional analysis of the ability of the glycosylation mutant receptors to inhibit forskolin-stimulated cAMP accumulation revealed that maximal inhibition of adenylate cyclase activity was similar in the mutant and wild-type receptors. The Asp2,3,6 mutant receptor had an unaltered IC50 value for carbachol while the IC50 value of the Gln2,3,6 mutant receptor was 2-fold higher than that of the wild-type receptor. These results indicate that N-glycosylation of the m2 mAChR is not required for cell surface localization or ligand binding and does not confer increased stability against receptor degradation. Furthermore, N-glycosylation of the m2 mAChR is not required for functional coupling of the m2 mAChR to inhibition of adenylate cyclase.

Muscarinic receptors in the mammalian heart.

In the mammalian heart, cardiac function is under the control of the sympathetic and parasympathetic nervous system. All regions of the mammalian heart are innervated by parasympathetic (vagal) nerves, although the supraventricular tissues are more densely innervated than the ventricles. Vagal activation causes stimulation of cardiac muscarinic acetylcholine receptors (M-ChR) that modulate pacemaker activity via I(f) and I(K.ACh), atrioventricular conduction, and directly (in atrium) or indirectly (in ventricles) force of contraction. However, the functional response elicited by M-ChR-activation depends on species, age, anatomic structure investigated, and M-ChR-agonist concentration used. Among the five M-ChR-subtypes M(2)-ChR is the predominant isoform present in the mammalian heart, while in the coronary circulation M(3)-ChR have been identified. In addition, evidence for a possible existence of an additional, not M(2)-ChR in the heart has been presented. M-ChR are subject to regulation by G-protein-coupled-receptor kinase. Alterations of cardiac M(2)-ChR in age and various kinds of disease are discussed.

Essential role of dynamin in internalization of M2 muscarinic acetylcholine and angiotensin AT1A receptors.

Most G protein-coupled receptors (GPCRs), including the M(1) muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M(2) mAChR and the angiotensin AT(1A) receptor (AT(1A)R) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M(2) mAChR and AT(1A)R in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4, 5-bisphosphate, strongly inhibits internalization of M(1) and M(2) mAChRs and AT(1A)Rs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M(1) mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M(1) mAChR internalization. In contrast, M(2) mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M(1) mAChR but also for M(2) mAChR and AT(1A)R internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.