The Calponin Family

The Calponin Family

Page updated 4/2/03

A family of actin binding proteins typified by smooth muscle specific protein which binds G and F-actin. Calponins are abundant proteins in smooth muscle and are the products of three distinct genes. The most abundant form is a-calponin (h1 or basic) is 292 amino acids (34kDa), b-calponin is 252 amino-acids in length have a deletion of amino-acids 217-256 of the same gene as a-calponin. Two other genes encode neutral calponin (h2 calponin) and acidic calponin are expressed at lower levels in smooth and non-muscle cells. Other more widely expressed calponin members include SM22, and transgelin. Calponin ironically has given its name to the CH or Calponin Homology domain present in many ABPs, despite the fact that calponin does not seem to bind actin through this region! (Gimona & Mital, 1998), but instead binds lipids (Bogatcheva & Busev, 1995; Fujii et al, 1995). The principle actin binding site is believed to exist (Mezgueldi et al, 1992; ) at a distinct region between the CH domain and the calponin repeats. Calponin is an abundant heat stable, basic protein in smooth muscle where it is present as a 34kDa species. In other tissues a more acidic isoform (acidic CaP) is present it is calcium independent but binds calmodulin in a sensitive manner. Binding of Ca²⁺/calmodulin inhibits actin binding. Phosphorylation also inhibits actin binding. Calponin binds tropomyosin another actin binding protein. Calponin binds many other proteins, many of which are also actin binding proteins (table 1) in addition to phospholipids (Bogatcheva & Busev, 1995).

Calponin Structure

Figure 1. The domain structure of calponin

The most celebrated feature of calponin is the so called Calponin Homology (CH) domain as mentioned previously. Calponins have just one single CH domain. Each CH domain is about 100 amino-acids in length and in the many actin binding proteins that are known to bind actin through CH domains, these are present as two dissimilar domains. When present singly as in calponin itself, it seems that the CH domain is insufficient to bind actin (like trying to pick up a pint with one hand while wearing boxing gloves!?) (Gimona & Mital, 1998). The fact that calponin does not bind actin through the CH domain is consistent with the observation that calponin competes neither with filamin (Panesenko & Gusev, 2001) or a-actinin for actin binding (Leinweber et al, 1999; Panesenko & Gusev, 2001), both of which bind through twin CH domains. The single CH domain in calponin is known to bind Ca²⁺/calmodulin, ERK, tropomyosin (Winder & Walsh, 1993). The structure of the single CH domain in calponin itself has overall similarity with the CH domains of those naturally arranged in tandem, such as in utrophin (Keep et al, 1999) or fimbrin (Goldsmith et al, 1997). The single calponin CH domain however has differs significantly possibly explaining why the single CH domain cannot bind actin (Bramham et al, 2002).

The regulatory domain (RD) contains the actin binding domain which has reported homology to troponin I (Takahashi et al, 1991). There are also sites for phosphorylation by PKC (Ser 175) and by CaM/kinase II.

Click-23

The calponin repeat domain at the C terminus is proposed to form WD40 like structures through which many interactions with other proteins is made. Secondary actin binding site may exist in the calponin repeats (Bonet-Kerrache & Mornet,1995), very possibly right next to the main site, at the start of repeat 1. Kolakowski et al, 1995; Tang et al, 1997). An actin binding site is reported to exist within the repeats. Recently, (Kranewitter et al, 2001) it has been shown that Unc-87 is an actin filament bundling protein composed of seven calponin repeats, these authors have dubbed the calponin repeat CLICK-23 (from the fact that the repeat is 23amino-acids in length and is Calponin Like).

Protein	Function	Binding site on Calponin	References
a-actinin	Strengthens the a-actinin-actin complex		Leinweber et al, 1999a
Actin	Modulates actin: myosin II ATPase activity.	Regulatory domain and	Mezgueldi,et al, 1992; 1995
Caldesmon	Myosin II binding/regulation
Caltropin	Myosin II binding/regulating	Regulatory domain	Wills et al, 1994
Calmodulin	Inhibits actin binding to regulatory domain in Ca²⁺.	Two sites, one in the CH (1-52) domain, the other in the RD (145-163)	Winder et al, 1993;
ERK	Extra-cellular regulatory kinase	CH domain	Leinweber et al, 1999b
Hsp70	Heat shock protein	7-144	Bogatcheva et al, 1999; Ma et al, 2000
Myosin II	Stimulated myosin II ATPase activity	146-176	Szymanski et al,1997; Lin et al,1993
Protein kinase C	Activates kinase activity	Calponin repeats (WD40)	Leinweber et al, 2000
Tropomyosin	Actin stability and myosin II regulation .	Two sites, one in the CH (1-52) domain, the other in the RD (145-163)	Takahashi et al, 1988; Vancompernolle et al, 1990;Childs et al, 1992; Mezgueldi,et al, 1995.
Tubulin	Microtubule binding and bundling? May also connect other cytoskeletal elements e.g. actin to microtubules.	Two sites, one in the actin and Calmodulin binding region 145-182, the other in the C terminal repeat region 183-292	Fuji et al, 1997; Fattoum et al, 2003

Table 1 Calponin-Binding proteins

Calponin Function
The function of the calponins has long been assumed to be to regulate the interact between actin filaments and myosin II in smooth muscle contraction and analogous interactions in non-muscle cells (Takahashi et al, 1986; Winder & Walsh 1993; El-Mezgueldi 1996; Gusev 2001). The consensus has been that h1 calponin inhibits the actin-activated ATPase of myosin II thereby regulating the interaction. However, the h1 gene has been knocked out in a mouse model (Yoshikawa et al, 1998) and the resulting phenotype has been a surprise. The h1 calponin was found not to be compensated by up-regulation of other calponins and the most obvious effect was on the bone (as is the knock out of another ABP ()). h1 calponin is highly expressed in osteoblasts and may act as a negative regulator of the bone making process. Analysis of the knock out mouse (Matthew et al, 2000), revealed that compared with wild type mouse there was no difference in Ca²⁺ sensitisation pathways but there was a clear increase in unloaded shortening velocity in K.O. mice muscle and that this could be overcome by adding back h1 calponin to the tissue. These findings are consistent with a role for calponin in the regulation of actin:myosin interaction but the study also showed a drastic reduction in the expression of actin (about 50%).

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