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Integrin αIIbβ3 mediates platelet aggregation and “outside-in” signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to αIIbβ3 function, αIIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified αIIb subunits were expressed with β3 in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25–50% of that observed when αIIbβ3 affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of αIIbβ3 also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72Syk and fibrinogen-dependent phosphorylation of pp125FAK, even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in αIIbβ3 function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of αIIbβ3 in platelets.

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Plasma growth hormone (GH) profiles are sexually differentiated in many species and regulate the sexdependence of peripubescent growth rates and liver function, including steroid hydroxylase cytochrome P450 expression, by mechanisms that are poorly understood. By use of an external pump to deliver to hypophysectomized rats pulses of rat GH of varying frequency and amplitude, a critical element for liver discrimination between male and female GH patterns was identified. Liver expression of the male-specific steroid 2a(orl6a)-hydroxylase P450, designated CYP2C11, was stimulated by GH at both physiological and nonphysiological pulse amplitudes, durations, and frequencies, provided that an interpulse interval of no detectable GH was maintained for at least 2.5 hr. This rmding suggests that hepatocytes undergo an obligatory recovery period after stimulation by a GH pulse. This period may be required to reset a GH-activated intracellular signaling pathway or may relate to the short-term absence of GH receptors at the hepatocyte surface after a cycle of GH binding and receptor internalization. These requirements were distinguished from those necessary for the stimulation by GH of normal male growth rates in hypophysectomized rats, indicating that different GH responses and, perhaps, different GH-responsive tissues recognize distinct signaling elements in the sexually dimorphic patterns of circulating GH.Episodic secretion is a general characteristic of many hormones, including pituitary-dependent hormones, pancreatic islet, and parathyroid hormones (1), and is often crucial to the triggering of hormone-dependent responses in target cells. For growth hormone (GH), secretion by the pituitary is not only intermittent, or pulsatile, but also the frequency of pulsation is sex-dependent. In many species, including the rat, chicken, and human (2-5), pituitary GH secretion is more frequent in females than in males. In the case of the female rat, a high pulse frequency results in GH present in circulation continuously, at levels 2 10-20 ng/ml of plasma. This situation contrasts with the intermittent presence of GH in plasma of male rats (2, 6). Studies in rats (7-10) and mice (11-13) have demonstrated that the expression of a number of sexually differentiated hepatic proteins, including cytochrome P450 (P450)-linked steroid hydroxylases and drugmetabolizing enzymes, is primarily determined by plasma GH profiles and only secondarily regulated by the gonadal hormones through their effects on the hypothalamo-pituitary axis and its control of GH secretion (14-16).The plasma GH profile in a male rat is characterized by a pulse of 200-250 ng of GH per ml every 3-4 hr followed by a 2-to 2.5-hr period when circulating GH is nearly undetectable (.2 nrg/ml). It is unclear, however, just what features in this pattern are recognized as male by the hepatocyte. This critical question is addressed by the present study, which uses a recently developed external pump apparatus (17) for generation of periodic GH pulses of various frequenc...

Integrin alphavbeta3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, alphavbeta3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins alphavbeta3 and alphavbeta5 from several species, to investigate the role of alphavbeta3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.

Integrin ␣ V ␤ 3 mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which ␣ V ␤ 3 is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, ␣ IIb ␤ 3 , has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of ␣ V ␤ 3 , a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single ␣ V integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated ␣ V ␤ 3 , but not to ␣ IIb ␤ 3 , in receptor and cell binding assays. ␣ V ␤ 3 affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K d ‫؍‬ 2.4 M), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K d ‫؍‬ 80 nM), with no change in receptor number. In contrast, ␣ V ␤ 3 in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of ␤ 3 cytoplasmic tails. Up-regulation of ␣ V ␤ 3 affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that ␣ V ␤ 3 is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.Integrins mediate cell adhesion and signaling during many developmental, physiological, and pathological processes (1-4). The ␤ 3 integrin family includes ␣ IIb ␤ 3 , often referred to as the fibrinogen receptor, and ␣ V ␤ 3 , the vitronectin receptor. ␣ IIb ␤ 3 is confined to megakaryocytes and platelets and is required for platelet aggregation through interactions with Arg-Gly-Asp (RGD)-containing adhesive ligands, including fibrinogen and von Willebrand factor (5). ␣ V ␤ 3 is more widely expressed in proliferating endothelial cells, arterial smooth muscle cells, osteoclasts, platelets, and certain subpopulations of leukocytes and tumor cells (6, 7). The list of cognate ligands for ␣ V ␤ 3 overlaps that for ␣ IIb ␤ 3 , but includes others, such as osteopontin, matrix metalloproteinase-2, and adenovirus penton base, which do not interact with ␣ IIb ␤ 3 (6, 8 -10). In the adult organism, ␣ V ␤ 3 has been implicated in processes ranging from wound healing to tumor angiogenesis (11), arterial restenosis (12), osteoporosis (13), tumor progression (14), and adenovirus internalization (8).One fundamental function of integrins is ligand binding, which in many cases is rapidly regulated by a process variously referred to as "integrin activation," "inside-out signaling," or "affinity/avidity modulation" (15-19). Integrin activation encompasses at least two events: 1) modulation of receptor affinity through conformational changes in the ␣␤ heterodimer and 2) modulation...