Ze Wang

Expression of myosin isoforms in smooth muscle cells in the corpus cavernosum penis

Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses approximately 4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17 isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (approximately 10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches approximately 23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses ∼4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (∼10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches ∼23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.

Mutagenesis Analysis of Functionally Important Domains within the C-terminal End of Smooth Muscle Caldesmon

The ability of chicken gizzard smooth muscle caldesmon (CaD) to inhibit actomyosin ATPase activity is due mainly to an inhibitory domain that resides within the C-terminal 67 amino acid residues of the CaD molecule. In the present study, a series of C-terminal truncation and internal deletion mutants of chicken gizzard smooth muscle CaD were systematically designed using a site-directed mutagenesis approach, and these mutant proteins were overexpressed in a baculovirus expression system. Analysis of actin binding and inhibition of actomyosin ATPase activity using these mutants identified a strong actin-binding motif of 6 amino acid residues (from Lys718 to Glu723), which also form the core sequence for CaD-induced inhibition of actomyosin ATPase. However, maximal inhibition by CaD requires the presence of residues 728-731, which are not associated with actin binding. Our data provide direct evidence for the requirement of actin binding to a specific region in CaD for CaD-induced inhibition of actin activation of smooth muscle myosin ATPase. Furthermore, our findings also show that the region between residues 690 and 717 is responsible for the weak inhibition of actomyosin ATPase and reveal that the inhibitory determinants located in the regions between residues 690 and 717 and residues 718 and 756 can function independently.

Alterations in the expression of the β-cytoplasmic and the γ-smooth muscle actins in hypertrophied urinary bladder smooth muscle

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle γ-actin, shows over 200% increase in the γ-actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in γ-actin, since the 2D gel electrophoresis does not distinguish γ-smooth muscle actin from γ-cytoplasmic actin. The bladder smooth muscle α-actin and the smooth muscle α-actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the β-cytoplasmic actin (37%) and a concomitant diminution in the β-cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.

CLEAVAGE OF CALDESMON AND CALPONIN BY CALPAIN : SUBSTRATE RECOGNITION IS NOT DEPENDENT ON CALMODULIN BINDING DOMAINS

The calmodulin binding proteins, caldesmon and calponin, are cleaved by both major isoforms of calpain in vitro. The patterns of fragments generated by each enzyme are essentially identical for a given substrate. Qualitatively, the cleavage pattern of each substrate is unchanged by the presence or absence of calmodulin suggesting that the interaction between calmodulin and these calmodulin-binding proteins does not alter substrate recognition by calpain. However, calmodulin (at microM concentrations) does have a small, but significant, inhibitory effect directly on calpain as evidenced by slower rates of cleavage of alpha-casein, a protein that does not bind calmodulin. Inhibition is more pronounced with mu-calpain (15-25%) than with m-calpain (6-10%). In order to demonstrate, unequivocally, that substrate recognition does not require an interaction between calpain and a substrates calmodulin-binding domain, recombinant, full-length caldesmon and a mutant lacking the calmodulin binding domain were tested as substrates for calpain in the presence and absence of calmodulin. Calpain produced similar cleavage patterns of the baculovirus expressed caldesmon and the truncated mutant. Competition experiments demonstrated that calpain does not discriminate between the truncated mutant and full length caldesmon. This suggests that substrate recognition by calpain was not altered significantly by the absence of the calmodulin-binding domain. Cleavage of a second calmodulin-binding protein, calponin was also examined. The rate of calponin cleavage was increased in the presence of calmodulin, an observation that is also inconsistent with any requirement for calpain to bind to its calmodulin-binding site. These results demonstrate that calmodulin-binding domains do not provide substrate recognition sites for calpains. It seems likely that the calmodulin-like regions of calpain function to bind calcium and to regulate enzyme conformation as required for activity and that they do not interact directly with most substrates.

Functional and Structural Relationship between the Calmodulin-binding, Actin-binding, and Actomyosin-ATPase Inhibitory Domains on the C Terminus of Smooth Muscle Caldesmon

Multiple functional domains responsible for calmodulin (CaM) binding and actin-binding/actomyosin ATPase inhibition are present in the region between residues 598–756 of the chicken gizzard smooth muscle caldesmon (CaD) molecule. To precisely localize these functional domains and to further elucidate the structural basis of these domains, we analyzed a series of purified mutants of chicken gizzard smooth muscle CaD generated by internal deletions of amino acid sequences and expression in a baculovirus expression system. Our results demonstrate that, in addition to a strong actin-binding site sequence between residues 718–723 (Wang, Z., and Chacko, S. (1996)J. Biol. Chem. 271, 25707–25714), two weak actin-binding motifs are present in the regions between residues 690–699 and 650–666. These weak actin-binding regions function independently and are associated with weak actomyosin inhibitory activity. Analysis of the CaM-binding sites A (residues 658–666) and B (residues 690–695), the major CaM-binding sites in the C-terminal region of CaD, provided direct evidence for the involvement of both CaM-binding sites in the CaM-mediated reversal of the inhibition of actomyosin ATPase activity by CaD and for the functional independence of the two CaM-binding sites. Furthermore, the sequences between residues 598–649, upstream of CaM-binding site A, and 700–717, downstream of CaM-binding site B, appear to have no effect on either actin-binding or CaM-binding. The data also suggest that both CaM-binding sites A and B structurally overlap or lie in close proximity to the adjacent weak actin-binding sites and weak actomyosin ATPase inhibitory determinants.

Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system

SummaryThree recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3′-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per 106 cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of Ca2+-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant.