The relevance of MRI blood-sensitive sequences in the diagnostic assessment of late-onset epilepsy.

OBJECTIVE: Sporadic cerebral amyloid angiopathy (CAA) is a degenerative brain small vessel disease of ageing resulting from progressive amyloid deposition in small arteries and arterioles of the cortex and leptomeninges. CAA may be diagnosed by the mean of Boston criteria, particularly with the use of the blood-sensitive T2* MRI sequences (GRE and SWI). Epileptic seizures have rarely been reported in CAA. PATIENTS AND METHODS: We describe two patients with late-onset unprovoked seizures due to CAA. A short literature review on this topic is presented. RESULTS: In our two patients with late-onset unprovoked seizures as the first manifestation of CAA, only GRE and SWI sequences lead to a correct diagnosis. In literature, only 15 patients with CAA presenting with seizures have been reported. In these subjects, data on seizures semiology and prognosis are scarce. CONCLUSIONS: Our report highlights the importance to perform blood-sensitive sequences in all subjects with LOE of otherwise unknown etiology, not to miss a diagnosis of CAA.

Functional convergence spasm: an unexpected finding in a patient with focal epilepsy.

OBJECTIVE: Convergence spasm is a clinical condition characterized by transient episodes of convergence, miosis and accommodation with strabismus and diplopia and it is usually a manifestation of a functional neurological disorder. We describe a patient with a challenging diagnosis of convergence spasm in the setting of occipital lobe epilepsy. CASE REPORT: A 52-year-old woman came for the assessment of focal epilepsy due to left occipital cortical dysplasia. During ocular motility tests, she presented with episodes of short duration (~10-30 seconds) of convergent strabismus. Neuropsychological evaluation showed a severe mixed anxiety-depressive disorder with a tendency toward somatization. RESULTS: Convergence spasm was recorded during video-EEG examination and no ictal activity was present. CONCLUSIONS: To our knowledge, no other report of functional convergence spasm in the context of focal epilepsy associated with cortical dysplasia has been described in literature.

Novel COL4A2 mutation causing familial malformations of cortical development.

OBJECTIVE: This article aimed to describe a novel COL4A2 mutation and the phenotypic features of two family members presenting with epilepsy and cortical development malformations. PATIENTS AND METHODS: The first patient is a 65-year-old woman with hematuria and adult-onset seizures. Brain MRI showed closed lip schizencephaly of right lateral sulcus associated with polymicrogyria of the surrounding cortex and areas of subcortical heterotopia. The second patient is a 40-year-old man, her son. He was born post-term with neonatal distress and psychomotor developmental delay with congenital left leg paresis and strabismus, as well as childhood-onset focal motor seizures. Brain MRI showed a right nucleus-capsular porencephalic cavitation with enlargement of the homolateral ventricle and a focal right occipital cortico-subcortical encephalomalacia. A small heterotopic band was also present in the frontal left subcortical region. RESULTS: We tested both patients with a NGS panel for genetic epilepsies, which evidenced a missense mutation in COL4A2 gene (c.2972G>A, causing the aminoacidic substitution Gly991Glu). CONCLUSIONS: The phenotypic spectrum associated with COL4A2 mutations has not been extensively described in the literature. Testing for COL4A mutations is indicated in patients with malformations of cortical development, particularly in the presence of familial conditions, even in the absence of porencephaly or early hemorrhagic strokes.

Cultural consultation as a model for training multidisciplinary mental healthcare professionals in cultural competence skills: preliminary results.

ACCESSIBLE SUMMARY: Lack of cultural competence in care contributes to poor experiences and outcomes from care for migrants and racial and ethnic minorities. As a result, health and social care organizations currently promote cultural competence of their workforce as a means of addressing persistent poor experiences and outcomes. At present, there are unsystematic and diverse ways of promoting cultural competence, and their impact on clinician skills and patient outcomes is unknown. We developed and implemented an innovative model, cultural consultation service (CCS), to promote cultural competence of clinicians and directly improve on patient experiences and outcomes from care. CCS model is an adaptation of the McGill model, which uses ethnographic methodology and medical anthropological knowledge. The method and approach not only contributes both to a broader conceptual and dynamic understanding of culture, but also to learning of cultural competence skills by healthcare professionals. The CCS model demonstrates that multidisciplinary workforce can acquire cultural competence skills better through the clinical encounter, as this promotes integration of learning into day-to-day practice. Results indicate that clinicians developed a broader and patient-centred understanding of culture, and gained skills in narrative-based assessment method, management of complexity of care, competing assumptions and expectations, and clinical cultural formulation. Cultural competence is defined as a set of skills, attitudes and practices that enable the healthcare professionals to deliver high-quality interventions to patients from diverse cultural backgrounds. Improving on the cultural competence skills of the workforce has been promoted as a way of reducing ethnic and racial inequalities in service outcomes. Currently, diverse models for training in cultural competence exist, mostly with no evidence of effect. We established an innovative narrative-based cultural consultation service in an inner-city area to work with community mental health services to improve on patients' outcomes and clinicians' cultural competence skills. We targeted 94 clinicians in four mental health service teams in the community. After initial training sessions, we used a cultural consultation model to facilitate 'in vivo' learning. During cultural consultation, we used an ethnographic interview method to assess patients in the presence of referring clinicians. Clinicians' self-reported measure of cultural competence using the Tool for Assessing Cultural Competence Training (n = 28, at follow-up) and evaluation forms (n = 16) filled at the end of each cultural consultation showed improvement in cultural competence skills. We conclude that cultural consultation model is an innovative way of training clinicians in cultural competence skills through a dynamic interactive process of learning within real clinical encounters.

The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses.

Internalization of the ligand/receptor complexes is a consequence of the activation of the gonadotropin receptors. Since the recycling or degradation of the internalized receptors results in the maintenance or loss of cell surface receptors respectively and this contributes to the loss of responsiveness, we hypothesized that the fate of the internalized receptors could be an important component of desensitization. We examined this hypothesis using the wild-type and mutants of the human LH (hLHR) receptors and follitropin receptors expressed in MA-10 and KK-1 cells respectively. The receptor mutants were chosen because they are routed mostly to a lysosomal degradation pathway whereas the wild-type receptors are recycled back to the surface. We have shown that agonist stimulation of cells expressing the mutant receptors results in a more pronounced loss of cell surface receptors and agonist responses than stimulation of cells expressing the wild-type receptors. We concluded that receptor recycling promotes the maintenance of cell surface receptors and preserves hormonal responsiveness. This property of the hLHR is likely to be physiologically important because there at least two hLHR-expressing tissues in pregnant women, the maternal corpus luteum and the fetal Leydig cells, where a loss of hormonal responsiveness induced by the elevated levels of human chorionic gonadotropin that occur during pregnancy is not desirable.

Mental health care in Rome.

AIM: To describe principles and characteristics of mental health care in Rome. METHOD: Based on existing data, service provision, number of professionals working in services, funding arrangements, pathways to care, user/carer involvement and specific issues are reported. RESULTS: After the Italian psychiatric reform of 1978, an extensive network of community-based services has been set up in Rome providing prevention, care and rehabilitation in mental health. A number of small public acute/emergency inpatient units inside general hospitals was created (median length of stay in 2002 = 8 days) to accomplish the shift from a hospital-based to a community-based psychiatric system of care. Some private structures provide inpatient assistance for less acute conditions (median length of stay in 2002 = 28 days), whilst the large Roman psychiatric hospital was closed in 1999. DISCUSSION: Whilst various issues of mental health care in Rome overlap with those in other European capitals, there also are some specific problems and features. During the last two decades, the mental health system in Rome has been successfully converted to a community-based one. Present issues concern a qualitative approach, with an increasing need to foresee adequate evaluation, especially considering mental health patients' satisfaction with services and economic outcomes.

Identification of two distinct structural motifs that, when added to the C-terminal tail of the rat LH receptor, redirect the internalized hormone-receptor complex from a degradation to a recycling pathway.

We show that most of the internalized rat LH receptor is routed to a lysosomal degradation pathway whereas a substantial portion of the human LH receptor is routed to a recycling pathway. Chimeras of these two receptors identified a linear amino acid sequence (GTALL) present near the C terminus of the human LH receptor that, when grafted onto the rat LH receptor, redirects most of the rat LH receptor to a recycling pathway. Removal of the GTALL sequence from the human LH receptor failed to affect its routing, however. The GTALL sequence shows homology with the C-terminal tetrapeptide (DSLL) of the beta2-adrenergic receptor, a motif that has been reported to mediate the recycling of the internalized beta2-adrenergic receptor by binding to ezrin-radixin-moesin-binding phosphoprotein-50. Addition of the DSLL tetrapeptide to the C terminus of the rat LH receptor also redirects most of the internalized rat LH receptor to a recycling pathway but, like the recycling of the human LH receptor, this rerouting is not mediated by ezrin-radixin-moesin-binding phosphoprotein-50. We conclude that most of the internalized rat LH receptor is degraded because its C-terminal tail lacks motifs that promote recycling and that two distinct, but homologous, motifs (DSLL at the C terminus or GTALL near the C terminus) can reroute the internalized rat LH receptor to a recycling pathway that is independent of ezrin-radixin-moesin-binding phosphoprotein-50.

Mutations of the second extracellular loop of the human lutropin receptor emphasize the importance of receptor activation and de-emphasize the importance of receptor phosphorylation in agonist-induced internalization.

Alanine scanning mutagenesis of the second extracellular loop of the human lutropin receptor (hLHR) showed that mutation of most of the residues present in this region either enhance or impair the internalization of agonist. A more complete analysis of four mutants, two that enhanced internalization (F515A and T521A) and two that impaired internalization (S512A and V519A), showed that the two mutants that impaired internalization also show a decrease in the sensitivity for agonist-induced cAMP accumulation, whereas the two mutants that enhanced internalization show an increase in the sensitivity for agonist-induced cAMP accumulation. None of these mutants had an effect on the agonist-induced phosphorylation of the hLHR, however. We conclude that, in contrast to the prevailing view of the relative importance of receptor phosphorylation in the internalization of G protein-coupled receptors, the phosphorylation of the hLHR is less important than the agonist-induced activation of the hLHR in the process of internalization.

Effect of activating and inactivating mutations on the phosphorylation and trafficking of the human lutropin/choriogonadotropin receptor.

The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact. Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.

Multiple distant amino acid residues present in the serpentine region of the follitropin receptor modulate the rate of agonist-induced internalization.

The amino acid sequences of the human (h) and rat (r) follitropin receptors (FSHR) are approximately 89% identical, but the half-time of internalization of agonist mediated by the rFSHR is approximately 3 times faster than that of the hFSHR. Chimeras of the hFSHR and the rFSHR showed that this difference in rate is dictated mostly by the serpentine domain. Further analysis identified six residues, two non-contiguous residues in the transmembrane helix 4 (Leu/Thr in the rFSHR and Met/Ile in the hFSHR), three non-contiguous residues in the third intracellular loop (Thr/Thr/Lys in the rFSHR and Ile/Asn/Arg in the hFSHR), and one in transmembrane helix 7 (Tyr in the rFSHR and His in the hFSHR) that are fully responsible for the difference in the rates of internalization of the hFSHR and the rFSHR.

The C-terminal tail of the rat lutropin/choriogonadotropin (CG) receptor independently modulates human (h)CG-induced internalization of the cell surface receptor and the lysosomal targeting of the internalized hCG-receptor complex.

The analysis of 21 progressive truncations of the C-terminal tail of the rat LH/CG receptor (rLHR) revealed the presence of a region delineated by residues 628-649 that, when removed, enhanced the degradation of the internalized human (h)CG. The analysis of these truncations also revealed the presence of a region delineated by residues 624-631 that, when removed, enhanced the rate of internalization of hCG. Since there is little overlap between these two regions, we conclude that the structural features of the rLHR that mediate internalization and degradation of the internalized hormone are different. Detailed analyses of cells expressing a truncation at Y637 (designated rLHR-t637) showed that the enhanced degradation of hCG observed in the these cells is due to an increase in the rate of transfer of the internalized hCG-rLHR complex from the endosomes to the lysosomes rather than to the enhanced dissociation of the hCG-rLHR complex in the lysosomes.

p38JAB1 binds to the intracellular precursor of the lutropin/choriogonadotropin receptor and promotes its degradation.

Using the C-terminal tail of the rat lutropin/choriogonadotropin receptor (rLHR) as "bait" in a yeast two-hybrid screen resulted in the identification of p38(JAB1) (a protein initially identified as a co-activator of c-Jun) as a putative rLHR binding partner. More recently p38(JAB1) has been shown to promote the degradation of a cyclin-dependent kinase inhibitor and to be a component of the COP9 signalosome. Microscopic localization of an epitope-tagged p38(JAB1) expressed in 293 cells revealed a punctuated perinuclear and cytosolic localization, while cell fractionation studies showed that most of the p38(JAB1) was in a high speed supernatant. Co-transfection of 293 cells revealed that p38(JAB1) binds to the immature 68-kDa precursor of the rLHR that resides in the endoplasmic reticulum and promotes its degradation. It does not appear to interact with the cell surface rLHR, however, and it does not affect its expression. When transfected into HeLa cells, p38(JAB1) potentiates the transcriptional activity of c-Jun, but co-transfection with rLHR prevents this effect. We conclude that p38(JAB1) interacts with the rLHR precursor and promotes its degradation. These results reveal a novel protein binding partner of the rLHR and are consistent with current views of the functions of p38(JAB1).

Seven non-contiguous intracellular residues of the lutropin/choriogonadotropin receptor dictate the rate of agonist-induced internalization and its sensitivity to non-visual arrestins.

The amino acid sequences of the human (h) and rat (r) lutropin/choriogonadotropin receptors (LHR) are 87% identical, but the rate of agonist-induced internalization of the hLHR is approximately 7 times faster than that of the rLHR. Chimeras of the hLHR and the rLHR showed that this rate is dictated by the serpentine domain and the cytoplasmic tail. Further mutational analysis identified seven residues, two adjacent residues in the second intracellular loop (Val/Gln in the rLHR and Ile/His in the hLHR), four non-contiguous residues in the third intracellular loop (Arg/Gln/Thr/Pro in the rLHR and Lys/Arg/Met/Thr in the hLHR), and one in the C-terminal tail (Leu in the rLHR and Phe in the hLHR), that are necessary and sufficient to impart the slow rate of internalization of the rLHR and the fast rate of internalization of the hLHR. The internalization of the rLHR and the hLHR display different sensitivities to the non-visual arrestins. Therefore, we also tested if the simultaneous exchange of these seven residues resulted in the exchange of this property. Since this was found to be the case, we propose that these seven residues identified here form a non-visual arrestin-binding site.

A dileucine-based motif in the C-terminal tail of the lutropin/choriogonadotropin receptor inhibits endocytosis of the agonist-receptor complex.

The rat lutropin/choriogonadotropin receptor (rLHR) is a member of the rhodopsin-like subfamily of G protein-coupled receptors that has two adjacent dileucine motifs in the C-terminal cytoplasmic tail. Here we show that simultaneous (L613,614,615,616A) or individual (L613,L614A or L615,616A) mutation of the two adjacent dileucine motifs to alanines results in mutants with enhanced rates of agonist-induced internalization. The L613,L614A mutation was much more effective in enhancing internalization than the L615,L616A mutation. Moreover, the L613A mutation was more effective than the L614A mutation. Because in the human LHR the residues equivalent to L613 and L614 of the rLHR are a phenylalanine and a leucine (F635 and L636), we also prepared mutants that exchanged these motifs. In the rLHR, an LL-to-FL exchange enhanced endocytosis, and in the human LHR, an FL-to-LL exchange impaired endocytosis. The internalization of rLHR-wt and rLRH-L613,L614A was inhibited by coexpression of the clathrin-binding domain of beta-arrestin. In fact, this manipulation reduced the enhanced rate of internalization of rLHR-L613,614A back to that of rLHR-wt. The L613,614A mutation does not affect the degradation of the internalized agonist or the membrane targeting of the nascent rLHR. The L615,616A mutation also did not affect degradation of the internalized agonist but impaired the membrane targeting of the nascent rLHR. We conclude that the dileucine-based motifs of the rLHR inhibit internalization and suggest that this inhibition may be due to an impairment in the binding of the rLHR to endogenous nonvisual arrestins.

Role of the rate of internalization of the agonist-receptor complex on the agonist-induced down-regulation of the lutropin/choriogonadotropin receptor.

The extent of agonist-induced down-regulation of the LH/CG receptor (LHR) in human kidney 293 cells transfected with the rat LHR (rLHR) is much lower than in two Leydig tumor cell lines (MA-10 and R2C) that express the rodent LHR endogenously. This difference can not be attributed to differences in the recycling of internalized receptors, or in the replenishment of new receptors at the cell surface. It can be correlated, however, with the half-life of internalization of the bound agonist, which is approximately 60 min in Leydig tumor cells and about 100 min in transfected 293 cells. To determine whether the rate of internalization of the bound agonist affects down-regulation, we compared these two parameters in 293 cells expressing four rLHR mutants that enhance internalization and three mutants that impair internalization. We show that all four mutations of the rLHR that enhanced internalization enhanced down-regulation, while only one of the three mutations that impaired internalization impaired down-regulation. In addition, cotransfections of 293 cells with the rLHR-wt and three constructs that enhanced internalization (G protein-coupled receptor kinase 2, beta-arrestin, and arrestin-3) increased down-regulation, while a related construct (visual arrestin) that had no effect on internalization also had no effect on down-regulation. We conclude that the rate of internalization of the agonist-LHR complex is the main determinant of the extent of down-regulation of the LHR.

The rate of internalization of the gonadotropin receptors is greatly affected by the origin of the extracellular domain.

Previous results from this laboratory have shown that human kidney (293) cells transfected with the rat follitropin receptor (rFSHR) internalize agonist (i.e. human follitropin, hFSH) at a rate similar to that of other agonist-G protein-coupled receptor complexes while 293 cells transfected with the rat lutropin/choriogonadotropin receptor (rLHR) internalize agonist (human choriogonadotropin, hCG) at a rate that is about 1 order of magnitude slower. Taking advantage of this difference and the high degree of homology between the rLHR and rFSHR, we have now used chimeras of these two receptors to begin to delineate structural features that influence their internalization. Analysis of six chimeras that exchanged only the transmembrane domains (designated FLF and LFL), only the COOH-terminal domains (FFL or LLF) or both domains (FLL or LFF) show that the origin of the extracellular domain is at least as important, if not more, than the origin of the transmembrane and COOH-terminal domains in determining the rate of internalization of the gonadotropin receptors. Thus, the rates of internalization of agonist internalization mediated by FFL, FLF, and FLL more closely resemble rFSHR than rLHR, while the rates of agonist internalization mediated by LLF, LFL, and LFF more closely resemble rLHR than rFSHR. The importance of the extracellular domain was also evident even upon overexpression of arrestin-3, a protein that enhances the rate of internalization of the wild-type receptors and chimeras by binding to their intracellular regions.

Comparative in-vitro activity of levofloxacin against isolates of bacteria from adult patients with community-acquired lower respiratory tract infections.

This study was conducted to evaluate the activity of levofloxacin in comparison with a range of antibacterial agents against recent isolates obtained consecutively from patients with community-acquired pneumonia (CAP) or acute exacerbation of chronic bronchitis (AECB) during the period 1995 to 1996. Susceptibility testing was carried out by either microdilution or the Etest, and interpreted according to NCCLS breakpoints. The activity of levofloxacin was compared with that of amoxycillin, amoxycillin-clavulanate, cefuroxime, cefixime, erythromycin, roxithromycin, clarithromycin, azithromycin, ofloxacin and ciprofloxacin. Clinically significant numbers of bacteria were recovered from 31 CAP and 94 AECB specimens. The predominant bacterial species in the CAP specimens were Streptococcus pneumoniae (21 isolates) and Haemophilus influenzae (four isolates). The AECB isolates mainly consisted of S. pneumoniae (38%), Moraxella catarrhalis (26%), H. influenzae (19%) and Pseudomonas aeruginosa (10%). The overall percentage susceptible of the isolates for each antibiotic was: amoxycillin, 64%; amoxycillin-clavulanate, 89%; cefuroxime, 87%; cefixime, 78%; erythromycin, 85%; roxithromycin, 87%; clarithromycin, 87%; azithromycin, 85%; ofloxacin, 95%; ciprofloxacin, 95%; and levofloxacin, 97%. The activities of levofloxacin and the other agents were also compared against 40 S. pneumoniae isolates, of which 20 were penicillin-non-susceptible, recovered from CAP and AECB specimens during the period 1994 to 1996. These strains were all susceptible to levofloxacin, but only 50% were susceptible to ciprofloxacin and 80% to ofloxacin. Twenty M. catarrhalis, 20 H. influenzae and 20 methicillin-susceptible S. aureus isolates were also all susceptible to levofloxacin. Furthermore, 20 community-acquired P. aeruginosa isolates showed similar percentage susceptible rates to levofloxacin and ciprofloxacin. These in-vitro results suggest that levofloxacin may be useful in the treatment of community-acquired lower respiratory tract infections.

Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor.

The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4alpha, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or G alpha t, a scavenger of G betagamma. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and G alpha t inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.

Mutations that induce constitutive activation and mutations that impair signal transduction modulate the basal and/or agonist-stimulated internalization of the Lutropin/Choriogonadotropin receptor.

Previous results from this laboratory suggested that the same active conformation of the lutropin/choriogonadotropin receptor (LHR) is involved in the stimulation of G proteins and in triggering the internalization of the bound agonist. We have now analyzed two naturally occurring, constitutively active mutants of the human LHR. These mutations were introduced into the rat LHR (rLHR) and are designated L435R and D556Y. Cells expressing rLHR-D556Y bind human choriogonadotropin (hCG) with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation does not affect the internalization of the free receptor, but it enhances the internalization of the agonist-occupied receptors approximately 3-fold. Cells expressing rLHR-L435R also bind hCG with normal affinity, exhibit a 47-fold increase in basal cAMP, and do not respond to hCG with a further increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors approximately 2- and approximately 17-fold, respectively. We conclude that the state of activation of the rLHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing rLHR-L435R is due to the fast rate of internalization of the bound hCG. The finding that membranes expressing rLHR-L435R (a system where internalization does not occur) respond to hCG with an increase in adenylyl cyclase activity supports this suggestion.

Signaling and phosphorylation-impaired mutants of the rat follitropin receptor reveal an activation- and phosphorylation-independent but arrestin-dependent pathway for internalization.

We have previously shown that the rat follitropin receptor (rFSHR) expressed in transfected cells becomes phosphorylated upon stimulation of the cells with agonist or a phorbol ester. Peptide mapping and mutagenesis studies have also shown that the agonist- or phorbol ester-induced phosphorylation of the rFSHR maps to Ser/Thr residues present in the first and third intracellular loops. The experiments presented herein were initially designed to test for the presence of additional phosphorylation sites on the second intracellular loop of the rFSHR. Analysis of two new mutants in which the two threonines in the second intracellular loop (rFSHR-2L) or the two threonines in the second intracellular loop and the seven Ser/Thr residues in the third intracellular loop (rFSHR-2L + 3L) were mutated showed that one or more of the two threonines in the second intracellular loop are phosphorylated in response to phorbol ester, but not in response to agonist stimulation. Since rFSHR-2L and rFSHR-2L + 3L displayed a reduction in agonist-induced signaling, two additional mutants (rFSHR-D389N and rFSHR-Y530F) were constructed in an attempt to better understand the relationship between the agonist-induced activation, phosphorylation, and internalization of the rFSHR. These point mutations impaired agonist-stimulated signal transduction and abolished agonist-induced phosphorylation. Co-transfection studies revealed that the phosphorylation of these mutants can be rescued by overexpression of G protein-coupled receptor kinase 2, but this increased phosphorylation only rescues the internalization of rFSHR-D389N. The internalization of both mutants could be rescued by overexpression of arrestin-3, however. Taken together, these results argue that the agonist-induced activation and phosphorylation of the rFSHR are not essential for internalization. while the interaction of the rFSHR with a nonvisual arrestin is essential for internalization.